Tools for the Study of Apoptosis, Cytotoxicity, and Cell Proliferation

www.roche-applied-science.com

Count on Roche Applied Science for superior products for the study of Apoptosis, Cytotoxicity, and Cell Proliferation. Our product portfolio includes reliable and easy-to-use kits, assays, and reagents that deliver accurate results for research in diverse applications.

Introduction Overview of Roche Products for Studying Apoptosis, Cytotoxicity, and Cell Proliferation Apoptosis Apoptosis Product Selection Guide Introduction to Apoptosis and Necrosis Caspase Activity Membrane Alterations DNA Fragmentation in Cell Populations DNA Fragmentation in Individual Cells Cytotoxicity Cytotoxicity Product Selection Guide Introduction to Cytotoxicity, Cell Proliferation, and Cell Viability Cytotoxicity in Cell Populations Cell Proliferation Cell Proliferation Product Selection Guide Metabolic Activity DNA Synthesis in Cell Populations DNA Synthesis in Individual Cells Ordering Information Products for Studying Apoptosis, Cytotoxicity, and Cell Proliferation Additional Products for the Study of Cells and Proteins

3 4 6 6 8 9 12 14 17 20 20 21 22 24 24 25 28 31 34 34 35

Gain a deeper understanding of cell death and proliferation processes and their regulation.

Differentiate between apoptosis and necrosis, study the cytotoxic or cytostatic effects of drugs or environmental pollutants on cells, determine what factors influence the rate and timing of cell proliferation, or investigate when and why cell death occurs these are just a few examples of how you can employ our kits and reagents in your research applications. Use this brochure to learn more about apoptosis, cytotoxicity, and cell proliferation and to determine the best product for your application needs. View helpful information including selection guides, product features, detection methods, suitable sample types, and typical results. Visit our Apoptosis Special Interest Site at
www.roche-applied-science.com/apoptosis and

Choose from our extensive product line of nonradioactive reagents, kits, and assays: Analyze various stages of the apoptotic
pathway, cytotoxicity, and cell proliferation

choose kits to measure DNA fragmentation, metabolic activity, or other specific parameters. Count on products that are broadly referenced in thousands of publications, time tested, and successfully used by researchers worldwide. Select from multiple detection formats light or fluorescence microscopy, flow cytometry, or ELISA with colorimetric or chemiluminescent substrates. Perform high-throughput screening assays
or analyze individual samples.

access a wealth of resources to support your research. Explore this comprehensive site to find technical tips, scientific and application information, references, Biochemica articles, and more. Download the Apoptosis, Cytotoxicity, and Cell Proliferation Manual to obtain further details about our products, protocols, application examples, and other valuable information.

Analyze many different sample types frozen or paraffin-embedded tissue sections, adherent or suspension cells, cell culture supernatants, individual cells, or cell populations.
View an Apoptotic Pathways Chart online at www.roche-applied-science.com/apoptosis by clicking on Scientific Information, and then Apoptotic Pathways.

Overview of Roche Products for Studying Apoptosis, Cytotoxicity, and Cell Proliferation

Apoptosis
If you are studying and you wish to detect using detection by then use Caspase Activity Caspase cleavage of cytokeratin 18 Western blot, flow cytometry, fluorescence microscopy, or light microscopy Fluorescence ELISA Fluorescence ELISA M30 CytoDEATH M30 CytoDEATH, Fluorescein page 9 9

Caspase 3 activity Caspases

Caspase 3 Activity Assay Homogeneous Caspases Assay, fluorimetric Anti-Poly (ADP-Ribose) Polymerase (PARP) Annexin-V-FLUOS Annexin-V-FLUOS Staining Kit Annexin-V-Alexa 568 Annexin-V-Biotin

10 11

Caspase cleavage of PARP

Western blot, immunoprecipitation, immunohistology Flow cytometry, fluorescence microscopy, or light microscopy

12

Membrane Alterations Phosphatidylserine in the outer layer of the plasma membrane

13 13 13 13 16 14 15 18 18 19 19 23

DNA Fragmentation

DNA fragments

Gel electrophoresis ELISA

Apoptotic DNA Ladder Kit Cell Death Detection ELISAPLUS Cell Death Detection ELISA In Situ Cell Death Detection Kit, AP In Situ Cell Death Detection Kit, POD In Situ Cell Death Detection Kit, Fluorescein In Situ Cell Death Detection Kit, TMR red

TUNEL

DNA Synthesis

BrdU incorporation

Colorimetric ELISA

Cellular DNA Fragmentation ELISA

Cytotoxicity
If you are studying and you wish to detect using detection by then use Plasma Membrane Damage Release of lactate dehydro- Colorimetric assay genase (LDH) Cytotoxicity Detection KitPLUS (LDH) Cytotoxicity Detection Kit (LDH) Cellular DNA Fragmentation ELISA page 22 23 23

DNA Synthesis

BrdU incorporation

Colorimetric ELISA

Cell Proliferation
If you are studying and you wish to detect using detection by then use Metabolic Activity Reduction of tetrazolium salts to formazan salts by viable cells ELISA Cell Proliferation Reagent WST-1* Cell Proliferation Kit I (MTT)* Cell Proliferation Kit II (XTT)* ATP Bioluminescence Assay Kit HS II ATP Bioluminescence Assay Kit CLS II Cell Proliferation ELISA, BrdU (colorimetric)* Cell Proliferation ELISA, BrdU (chemiluminescent)* BrdU Labeling and Detection Kit III (POD)* page 25 26 26 27 27 28 29

ATP levels

Bioluminescence

DNA Synthesis

BrdU incorporation

Colorimetric ELISA Chemiluminescence ELISA Colorimetric ELISA

30 31 31 32 33

Fluorescence micros- BrdU Labeling and copy or flow cytometry Detection Kit I (Fluorescein) Light microscopy BrdU Labeling and Detection Kit II (AP)

Fluorescence micros- In Situ Cell Proliferation copy or flow cytometry Kit, FLUOS Multiple methods
* This product can also be used to study cytotoxicity.

Anti-BrdU Antibodies

Apoptosis Product Selection Guide

Start
DNA fragmentation, qualitative

Apoptotic DNA Ladder Kit

Caspase activity, indirect

11 835 246 001

Anti-Poly (ADP-Ribose) Polymerase (PARP)

11 835 238 001

using detection by western gel blot electrophoresis

Apoptosis

western blot or gel electrophoresis using detection by ELISA

If you are studying cell populations single cells

using detection by colorimetric fluorimetric ELISA ELISA

FACS or light fluorescence microscopy microscopy

using detection by

DNA fragmentation, quantitative

DNA fragmentation

Cell Death Detection ELISAPLUS

11 774 425 001 11 920 685 001 11 585 045 001

In Situ Cell Death Detection Kit, TMR red In Situ Cell Death Detection Kit, Fluorescein
Caspase activity, indirect

12 156 792 910 11 684 795 910

BrdU incorporation in DNA fragments, quantitative

Cellular DNA Fragmentation ELISA

Caspase activity, quantitative

M30 CytoDEATH, Fluorescein M30 CytoDEATH 12 012 952 001 03 005 372 001 12 236 869 001
Membrane modification

12 156 857 001 12 140 322 001 12 140 349 001 03 703 126 001 11 828 681 001 11 858 777 001 11 988 549 001

Caspase 3 Activity Assay Homogeneous Caspases Assay, fluorimetric

Annexin-V-Alexa 568 Annexin-V-FLUOS Annexin-V-FLUOS Staining Kit

Follow the selection guide below to determine the appropriate Roche Applied Science product for the study of apoptosis to meet your needs. See page 20 for products to study cytotoxicity and page 24 for products to study cell proliferation. If you need additional help, please visit
www.roche-applied-science.com/apoptosis
DNA fragmentation

In Situ Cell Death Detection Kit, TMR red In Situ Cell Death Detection Kit, Fluorescein
Caspase activity, indirect

12 156 792 910 11 684 795 910

Apoptosis

M30 CytoDEATH, Fluorescein M30 CytoDEATH

12 156 857 001 12 140 322 001 12 140 349 001

in in cell cultures, tissue blood cells, sections etc.

FACS or fluorescence microscopy using detection by light microscopy

DNA fragmentation

In Situ Cell Death Detection Kit, POD In Situ Cell Death Detection Kit, AP
DNA fragmentation Caspase activity, indirect

11 684 817 910 11 684 809 910 12 140 322 001 12 140 349 001

In Situ Cell Death Detection Kit, POD In Situ Cell Death Detection Kit, AP
Caspase activity, indirect

11 684 817 910 11 684 809 910 12 140 322 001 12 140 349 001 11 828 690 001

M30 CytoDEATH

M30 CytoDEATH
Membrane modification

Annexin-V-Biotin

Introduction to Apoptosis and Necrosis

Cell death can occur by either of two distinct mechanisms necrosis or apoptosis.
Necrosis (sudden or accidental cell death) is

Caspase Activity
Caspases (cysteinyl-aspartic acid proteases) are a family of proteases that cleave vital intracellular proteins and drive the progression of apoptosis. Caspases (expressed as zymogenes) are activated by different inducers. Once activated, a single caspase activates a cascade of other caspases. Caspases are expressed very early in apoptosis (see Figure 1) as well as in later stages of apoptosis.

the pathological process that occurs when cells are exposed to a serious physical or chemical injury.
Apoptosis (programmed cell death) is a natural biological phenomenon by which unwanted cells are eliminated during development and other physiological processes. The sequence of events that a cell undergoes in the course of apoptosis is highly regulated and regimented. Several of these physiological events can be used to monitor the progression of apoptosis (Figure 1). Different methods of apoptosis induction can proceed via different pathways, necessitating the use of several assay methods for confirmation.

Membrane Alterations
In normal cells, phosphatidylserine is found only on the inner leaflet of the plasma membrane. In apoptotic cells, some of the phosphatidylserine migrates to the outer leaflet and can be detected by AnnexinV (a phospholipid-binding protein with high affinity for phosphatidylserine). When cell membranes of necrotic cells break, the phosphatidylserine on the inner leaflet is also exposed. However, these cells are unable to exclude a vital dye, such as propidium iodide, enabling discrimination between apoptotic and necrotic cells.

Apoptosis

Roche Applied Science offers products for the investigation and characterization of cell death through the study of: Caspase activity Membrane alterations DNA fragmentation

DNA Fragmentation
As apoptosis progresses (Figure 1), activated endonucleases break down genomic DNA between nucleosomes, generating mono- and oligonucleosomal DNA fragments (Figure 21 on page 16). This biochemical hallmark of apoptosis is an irreversible event and commits the cell to die.

Apoptosis induction by TNF-/Actinomycin D Caspase activity initiated FLICE activation plays a critical role in the apoptotic signaling cascade Formation of cytochrome c/apaf-1 complex Decreasing mitochondrial potential Additional caspase activity Several caspases are recruited to carry out a critical proteolytic function Expression of apoptosis-related proteins. The status of several proteins is indicative of apoptosis Membrane alterations Cell membrane composition changes, partially as a signal to encourage the endocytosis of the apoptotic cells

Figure 1: Typical apoptosis progression. Sequential physiological events that are known to occur in HeLa cells undergoing apoptosis after induction with TNF-/Actinomycin D.

Morphological changes DNA fragmentation Destruction of the genetic material is an irreversible step in apoptosis

Time

Caspase Activity

M30 CytoDEATH M30 CytoDEATH, Fluorescein

Detect early apoptosis with an antibody

During apoptosis, vital intracellular proteins are cleaved. The proteases that mediate this process are called caspases (cysteinylaspartic acid proteases). Caspases are expressed as zymogenes, which are activated by different apoptosis inducers. Once activated, a single caspase activates a cascade of caspases. The mouse monoclonal M30 CytoDEATH antibody recognizes a specific (formalin-resistant) caspase-cleavage epitope in human cytokeratin 18 (CK 18) cytoskeletal protein in apoptotic cells. The antibody does not bind uncleaved native cytokeratin 18 of normal cells. Thus, the M30 CytoDEATH or M30 CytoDEATH, Fluorescein antibodies are valuable tools for the easy and reliable determination of very early apoptotic events in single cells and tissue sections. Detect early apoptosis in epithelial cells and tissue sections with high sensitivity (Figures 2 and 4). Determine caspase activity even in formalin-fixed, paraffin-embedded tissue (Figure 4). Choose from two antibody formats to meet your specific application needs. Easily perform fluorescence (Figure 2) and FACS (Figure 3) analysis with M30 CytoDEATH, Fluorescein. Use in dual-labeling applications, for example, in combination with the In Situ Cell Death Detection Kit, TMR red (TUNEL technique, see pages 17 - 19). Save time with a convenient, easy-to-use protocol.
Sample material: adherent cells, routinely fixed tissue sections,
Figure 2: Detection of apoptosis in HeLa cells treated with TNF- and Actinomycin D, using M30 CytoDEATH, Fluorescein.

Apoptosis

Figure 3: FACS analysis of HeLa cells, using M30 CytoDEATH, Fluorescein, after apoptosis induction with TNF-/Actinomycin D. White: Untreated control cells. Red: Cells treated with TNF- and Actinomycin D.

paraffin-embedded tissue sections, and cryostat sections.

Product M30 CytoDEATH M30 CytoDEATH, Fluorescein Cat. No. 12 140 322 001 12 140 349 001 12 156 857 001 Pack Size 50 tests 250 tests 250 tests Figure 4: Human colon tissue demonstrating positive staining for caspase-cleaved cytokeratin 18. Detection of apoptosis with M30 CytoDEATH, anti-mouse-biotin, streptavidin-HRP, and AEC (substrate). Counterstained with hematoxylin. M30 staining (brown) appears in the cytoplasm of the cell. This assay works well in combination with TUNEL reactions.

Caspase Activity

Caspase 3 Activity Assay

Detect caspase 3 activity with an ELISA

The activation of caspase 3 plays a key role in the apoptotic process and occurs in early apoptosis. The Caspase 3 Activity Assay is a fluorimetric immunosorbent enzyme assay (FIENA) for the specific and quantitative in vitro determination of caspase 3 activity (Figures 6 and 7). Specifically detect caspase 3 activity. Detect only natural and recombinant human caspase 3 activity in research samples.
Apoptosis

Detect low levels of caspase 3 activity even in populations where as little as 5% of cells are apoptotic. Detect and obtain semiquantitative data from samples undergoing the early stages of apoptosis. Perform kinetic assays. Lysates are stable for up to six months, allowing studies at multiple time points. Screen multiple samples simultaneously. Take advantage of a convenient 96-well microplate format when screening multiple samples (Figure 5).
Sample material: lysates of apoptosis-induced cells; recombi-

Figure 6: Use of the Caspase 3 Activity Assay to measure apoptosis induction in U-937 cells following exposure to various concentrations of camptothecin (an apoptosis-inducing antibiotic). Lysates were analyzed with the Caspase 3 Activity Assay and with Annexin-V-FLUOS. Caspase 3 activity is proportional to the percentage of apoptotic cells.

nant caspase 3 (synonyms: CPP32, YAMA, and Apopain).

Figure 5: Schematic showing the principle of the Caspase 3 Activity Assay. Ac-DEVD-AFC = Acetyl-Asp-Glu-Val-Asp-7-amido-4-trifluoromethylcoumarin. AFC = 7-amido-4-trifluoromethyl-coumarin Product Caspase 3 Activity Assay Cat. No. 12 012 952 001 Pack Size 1 kit (96 tests)

Figure 7: Use of the Caspase 3 Activity Assay to measure apoptosis induction in U-937 cells following exposure to 4 g/ml camptothecin for different time intervals at 37C. Lysates were analyzed for caspase 3 activity, and fluorescence was plotted versus time. Additionally, an aliquot of the same cells was analyzed for Annexin-V binding in parallel. Result: Figures 6 and 7 demonstrate that Caspase 3 activity and Annexin-V binding correlate very closely in both dose-response and kinetic studies.

10

HTS

Homogeneous Caspases Assay, fluorimetric

Detect multiple caspase activities with an ELISA

Measure a broad spectrum of caspase activity in multiple samples with this quantitative high-throughput assay. Detect several types of activated caspases in a single
assay.

Easily obtain results from this one-plate assay by performing experiments directly in a culture plate or a microplate (MP) (Figure 8). Use a convenient, one-step fluorimetric assay - no washing step is needed. Save time with an assay that takes just 2.5 hours. Simultaneously analyze large numbers of samples in this high-throughput format (Figures 9 and 10).
Sample material: cultured cells of human or animal origin.

Figure 9: Use of the Homogeneous Caspases Assay, fluorimetric to detect apoptosis induction in U-937 cells following exposure to camptothecin. U-937 cells were seeded in two densities, and apoptosis was induced with 4 g/ml camptothecin. The activity of caspases was quantified at different time points.

Apoptosis

Figure 8: Schematic showing the principle of the Homogeneous Caspases Assay, fluorimetric. DEVD-R110 = Asp-Glu-Val-Asp-Rhodamine 110 R110 = Rhodamine 110 Product Homogeneous Caspases Assay, uorimetric Cat. No. 03 005 372 001 Pack Size

Figure 10: Use of the Homogeneous Caspases Assay, fluorimetric to detect apoptosis induction in U-937 cells exposed to different concentrations of camptothecin for 4 hours at 37C. The cells were analyzed for caspase activity with the kit, then standardized values were plotted versus concentration. Result: Figure 9 illustrates the excellent linearity of kinetic studies and Figure 10 the sensitivity of dose-response studies using the Homogeneous Caspases Assay.

100 tests (96-well) 400 tests (384-well) 12 236 869 001 1,000 tests (96-well) 4,000 tests (384-well)

HTS

= Suitable for high-throughput screening.

11

Caspase Activity

Membrane Alterations

Anti-Poly (ADP-Ribose) Polymerase

Detect early apoptosis-induced proteolysis

Annexin-V Conjugates
Detect apoptosis-induced loss of plasma membrane asymetry and distinguish apoptosis from necrosis

Apoptosis

Poly (ADP-Ribose) Polymerase (PARP) is a nuclear enzyme involved in DNA repair. In many cell types, an early biochemical event in apoptosis is the proteolytic cleavage of PARP by a caspase at a highly conserved cleavage site. Use Anti-Poly (ADP-Ribose) Polymerase (PARP) antibody to detect early apoptotic changes through determination of PARP proteolysis in cell extracts. The appearance of a large (approximately 85 kD) cleavage fragment is indicative of caspase proteolytic activity (Figure 11). Use in immunoprecipitation, western blot, and immunohistochemistry. Detect early apoptosis by determining caspase cleavage of PARP. Use in dual-labeling applications, for example, in combination with the In Situ Cell Death Detection Kit, TMR red (TUNEL technique, pages 17-19). Detect full-length PARP, as well as large PARP fragments generated by caspases.
Sample material: samples of primate or rodent

In normal cells (Figure 12A), the distribution of phospholipids is asymmetric, with the inner membrane containing anionic phospholipids (such as phosphatidylserine) and the outer membrane having mostly neutral phospholipids. In apoptotic cells (Figure 12B) however, the amount of phosphatidylserine (PS) on the outer surface of the membrane increases, exposing PS to the surrounding liquid.
Annexin-V, a calcium-dependent phospholipid-bind-

ing protein, has a high affinity for PS. Although it will not bind to normal living cells, Annexin-V will bind to the PS exposed on the surface of apoptotic cells (Figures 13-15).
A B

origin.
1 2 3 4
116 kD (native PARP) 85 kD (cleaved PARP)

Figure 11: Detection of PARP cleavage fragments following apoptosis induction of MCF-7 cells. Extracts from treated and untreated cells were analyzed on western blot, followed by indirect immunodetection with Anti-PARP. Results: Anti-PARP recognizes intact (116 kD) and cleaved (85 kD) PARP. Lane 1: Untreated control cells Lane 2: Cells treated with 100 ng/ml doxorubicin for 24 hours Lane 3: Cells treated with 1 mg/ml methotrexate for 24 hours Lane 4: Cells treated with 1 mg/ml cytarabin for 24 hours Product Anti-Poly (ADPRibose) Polymerase Cat. No. 11 835 238 001 Pack Size 100 l

Figure 12: Detection of surface morphology changes during apoptosis. An exogenously added molecule specific for phosphatidylserine, such as Annexin-V-FLUOS, will bind to phosphatidylserine on the outer membrane of apoptotic cells, but cannot react with the phosphatidylserine of normal cells.
Normal Cells Annexin-V staining Propidium iodide staining Apoptotic Cells + Necrotic Cells + +

Table 1: Distinguishing apoptosis from necrosis using Annexin-V and propidium iodide.

12

Detect apoptosis using flow cytometry or microscopy with your choice of Annexin-V conjugates

Rapidly quantify apoptotic cells in cell suspensions by flow cytometry with Annexin-V-FLUOS (Figure 15). Distinguish necrotic cells from apoptotic cells by using the Annexin-V-FLUOS Staining Kit, which includes propidium iodide (Table 1, Figures 13 and 15). Analyze cell cultures or tissues by fluorescence microscopy using the FLUOS or Alexa 568 conjugates (Figures 13 and 14). Identify individual apoptotic cells by light microscopy with Annexin-V-Biotin.
Sample material: cell lines or freshly isolated cells.
Figure 13: Discrimination between apoptotic and necrotic U-937 cells treated with camptothecin. Late-stage apoptosis detected with Annexin-V-FLUOS (green), and counterstained with propidium iodide (necrotic cells).
Apoptosis

Figure 14: Discrimination between apoptotic and necrotic U-937 cells treated with camptothecin (CAM) and stained with Annexin-VAlexa 568 (red) and BOBO-1 (green).

Product Annexin-V-FLUOS Annexin-V-FLUOS Staining Kit (includes propidium iodide) Annexin-V-Alexa 568 Annexin-V-Biotin

Cat. No. 11 828 681 001 11 858 777 001 11 988 549 001 03 703 126 001 11 828 690 001

Pack Size 250 tests 1 kit (50 tests) 1 kit (250 tests) 250 tests 250 tests

Figure 15: Apoptotic and necrotic U-937 cells identified in FACS analysis after staining with Annexin-V-FLUOS and propidium iodide (PI). Cells were cultivated for 4 hours in the presence (lower row) or absence (upper row) of 4 g/ml camptothecin (CAM).
(A) Single-parameter analysis with Annexin-VFLUOS (B) Single-parameter analysis with propidium iodide (C) Dual-parameter analysis with Annexin-VFLUOS (FL1) and propidium iodide (FL2)

13

DNA Fragmentation in Cell Populations

HTS

Cell Death Detection ELISAPLUS

Distinguish apoptotic cells from necrotic cells with a onestep ELISA

Use the Cell Death Detection ELISAPLUS (CDDE PLUS) to analyze histone-associated DNA fragments (mono- and oligonucleosomes) (Figures 16 and 21), which are known to be present in the cytoplasm of cells undergoing apoptosis (Figures 17 and 19).
Apoptosis

Obtain reproducible relative quantification of cell death that correlates well with the DNA ladder method. Perform high-throughput analysis of cell death with a one-step ELISA that processes hundreds of cell samples in parallel, saving time and effort (Figure 16). Discriminate between cells undergoing apoptosis and those undergoing necrosis using one sample (Figure 17). Achieve high sensitivity by detecting apoptosis in as few as 600 cells (Figure 18). Choose the improved Cell Death Detection ELISAPLUS and obtain all the benefits of the Cell Death Detection ELISA with the added advantages of fewer steps, a shorter assay time, and an ABTS Stop Solution.
Sample material: cytoplasmic fractions (lysates) of cell lines,
Figure 17: Sample preparation steps for the Cell Death Detection ELISAPLUS.

cells ex vivo, cell culture supernatants, and serum or plasma.

Figure 18: Use of the Cell Death Detection ELISAPLUS to detect the increase in nucleosome concentration in the cytoplasmic fraction of U-937 cells treated with an apoptosis-inducing antibiotic (CAM). Different concentrations of U-937 cells were incubated in the absence or presence of 2 g/ml camptothecin (CAM) for 4 hours at 37C. Samples of culture supernatant and cell lysate were analyzed for nucleosome content. Result: The CDDE PLUS can clearly detect apoptosisrelated nucleosomes in as few as 600 cells.

Figure 16: Schematic showing the principle of the CDDE PLUS.

14

Utilize the Cell Death Detection ELISAPLUS to perform relative quantification of samples and compare the effects of apoptosisinducing agents on a population of cells. Generate numerical measurements of nucleosomal particles (Figures 18 and 19) rather than a qualitative banding pattern on a gel.
Figure 19: Use of the Cell Death Detection ELISAPLUS to detect dose- dependent induction of apoptosis in U-937 cells. U-937 cells (104 cells/ well, in 200 l) were incubated with different concentrations of camptothecin (CAM) for 4 hours at 37C. Before and after lysis, cells were centrifuged and the supernatant was analyzed. Results were plotted as dose versus response. Lysate (), Supernatant (), Enrichment factor of the lysate () Result: The ELISA data demonstrates that amounts of cytoplasmic oligonucleosomes (an indicator of apoptosis) increase as CAM concentration increases. Cell culture supernatants removed from the cells after treatment (but before lysis) gave no signal, indicating that there are no necrotic cells during the treatment. Product Cell Death Detection ELISAPLUS Cat. No. 11 774 425 001 11 920 685 001 Pack Size 96 tests 10 x 96 tests

Apoptosis

HTS

= Suitable for high-throughput screening.

Also available: The first-generation Cell Death Detection ELISA is a three-step assay for analyzing cell death using a 96-well microplate format.
Product Cell Death Detection ELISA Cat. No. 11 544 675 001 Pack Size 96 tests

15

DNA Fragmentation in Cell Populations

Apoptotic DNA Ladder Kit

Detect apoptosis by DNA ladder analysis using gel electrophoresis

Cleavage of genomic DNA into histone-associated DNA fragments (mono- and oligonucleosomes) is a biochemical hallmark of cells undergoing apoptosis (Figure 21). Upon purification and gel electrophoresis, these DNA fragments produce a distinctive ladder pattern (Figure 22). The purification method employed in the kit is much faster than other DNA purification methods (e.g., phenol/chloroform extraction, DNA precipitation). Purified DNA may be mixed directly with gel loading buffer and analyzed on an agarose gel. The Apoptotic DNA Ladder Kit can be used to:
Apoptosis

Figure 22: Demonstration of a typical DNA ladder in apoptotic cells, visualized with the Apoptotic DNA Ladder Kit. Lane Identification: M Size marker Control cells without camptothecin + Cells treated with camptothecin C Positive control from the kit

Purify DNA from cell samples in less than 20 minutes. Simplify DNA extraction by eliminating organic extractions and DNA-precipitation steps. Compare your results to the kits supplied control (fragmented DNA purified from lyophilized apoptotic U-937 cells), simplifying data interpretation.
Sample material: whole blood or cultured cells.

Figure 21: Overview of the mechanism of DNA fragmentation and the appearance of the DNA ladder. Product Apoptotic DNA Ladder Kit Cat. No. 11 835 246 001 Pack Size 1 kit (20 tests)

16

DNA Fragmentation in Individual Cells

TUNEL Assays for detection of apoptosis

Extensive DNA degradation, one of the hallmarks of apoptosis, may generate double-stranded, low molecular weight DNA fragments (mono- and oligonucleosomes) as well as single strand breaks (nicks) in high molecular weight DNA. Those DNA strand breaks can be detected by enzymatic labeling of the free 3'-OH termini with modified nucleotides. This end-labeling method, TUNEL (Terminal deoxynucleotidyl transferase [TdT]mediated dUTP Nick End Labeling), enables the highly sensitive detection of apoptosis in tissue and single cells (Figure 23). The use of fluorescein-dUTP to label the DNA strand breaks allows the detection of the incorporated nucleotides directly with a fluorescence microscope or a flow cytometer. Direct labeling with fluorescein-dUTP offers several other advantages. Direct labeling (Figure 24A) produces less nonspecific background with sensitivity equal to indirect labeling (Figure 24B) and, thus, is as powerful as the indirect method in detecting apoptosis. Furthermore, the fluorescence may be converted into a colorimetric signal if an anti-fluorescein antibody conjugated with a reporter enzyme (see Table 2 on page 18) is added to the sample.

In Situ End Labeling (TUNEL) (template independent)

Figure 23: Schematic illustration of In Situ End Labeling of DNA (TUNEL).

Apoptosis

Figure 24: Comparison of direct (A) and indirect (B) labeling of DNA strand breaks in apoptotic cells. (Data provided by R. Sgonc, University of Innsbruck, Austria). Result: Direct labeling is as sensitive as indirect labeling, but produces less nonspecific background.

17

DNA Fragmentation in Individual Cells

In Situ Cell Death Detection Kits

Use versatile TUNEL-based kits to detect apoptosis in single cells with high sensitivity

Method/Roche product

Label

Indirect (secondary) detection system None (direct detection)

Analysis by

Choose from four TUNEL labeling assays to: Achieve maximum sensitivity with minimal background. Detect low levels of apoptosis in tissue and single cells. Measure and quantify cell death with your choice of detection formats - choose kits for flow cytometry, fluorescence microscopy, or light microscopy.
Sample material: cells in suspension, cytospin and

FluoresceinIn Situ Cell Death Detection dUTP Kit, Fluorescein TMR-dUTP In Situ Cell Death Detection Kit, TMR red FluoresceinIn Situ Cell Death Detection dUTP Kit, AP FluoresceinIn Situ Cell Death Detection dUTP Kit, POD

Flow cytometry Fluorescence microscopy Fluorescence microscopy Light microscopy Light microscopy

None (direct detection) Anti-Fluorescein-AP Anti-Fluorescein-POD

Apoptosis

cell smear preparations, adherent cells grown on slides, and frozen and paraffin-embedded tissue sections.

Table 2: Choose from four different kits for the enzymatic labeling of DNA and the secondary detection systems required.

In Situ Cell Death Detection Kit, AP

Label cells with fluorescein-dUTP, and detect with anti-fluorescein-alkaline phosphatase (AP). Detect apoptotic cells via light microscopy using a colorimetric substrate.

TUNEL Assays for light microscopy

In Situ Cell Death Detection Kit, POD

Label cells with fluorescein-dUTP, and detect with anti-fluorescein-peroxidase (HRP, POD, horseradish peroxidase). Detect apoptotic cells via light microscopy using a colorimetric substrate (Figure 26).

Figure 26: Detection of apoptotic cells by TUNEL and peroxidase staining in rabbit endometrium. The tissue section was prepared and assayed with the In Situ Cell Death Detection Kit, POD, counterstained with hematoxylin, and viewed under a light microscope. Brown color indicates apoptotic nuclei as visualized using DAB substrate. Product In Situ Cell Death Detection Kit, AP Cat. No. 11 684 809 910 11 684 817 910 Pack Size 50 tests 50 tests

Figure 25: Schematic showing the principle of the In Situ Cell Death Detection Kits, AP and POD.

In Situ Cell Death Detection Kit, POD

18

In Situ Cell Death Detection Kit, Fluorescein

Label cells with fluorescein-dUTP. Detect apoptotic cells via fluorescence microscopy or flow cytometry (Figures 28-30).

TUNEL Assays for fluorescence microscopy and flow cytometry

In Situ Cell Death Detection Kit, TMR red

Label cells with tetramethylrhodamine-dUTP. Detect apoptotic cells via fluorescence microscopy or flow cytometry (Figure 31).

Figure 29: Cell suspension stained with the In Situ Cell Death Detection Kit, Fluorescein. U-937 cells induced with 4 g/ml camptothecin show positive staining of apoptotic nuclei. Note: This figure shows a high number (>80%) of apoptotic cells. To avoid detecting cells that are undergoing secondary necrosis, analyze cells earlier in the process after induction of apoptosis.

Apoptosis

Figure 27: Schematic showing the principle of the In Situ Cell Death Detection Kits, Fluorescein and TMR red. A B

Figure 30: Use of the In Situ Cell Death Detection Kit, Fluorescein to detect apoptotic cells (green) by fluorescence microscopy. A tissue section of rabbit endometrium was prepared and stained with the kit. This figure displays positive-staining nuclei (bright green) and autofluorescence in the surrounding healthy tissue. Figure 28: Detection of apoptotic cells by flow cytometry using the In Situ Cell Death Detection Kit, Fluorescein. HL60 cells were cultured in the absence [A] or presence [B] of 2 g/ml camptothecin for 3 hours at 37C. Control for autofluorescence of cells, without incubation with Label or Enzyme Solution Negative control, incubated with Label Solution, in the absence of terminal transferase Test sample, incubated with TUNEL reaction mixture Product In Situ Cell Death Detection Kit, Fluorescein In Situ Cell Death Detection Kit, TMR red Cat. No. 11 684 795 910 12 156 792 910 Pack Size 50 tests 50 tests Figure 31: Use of the In Situ Cell Death Detection Kit, TMR red to detect apoptotic cells (red) by immunohistochemical staining. Tissue from rabbit endometrium was assayed with the kit and viewed under a fluorescence microscope. Apoptotic nuclei stain bright red; limited fluorescence is visible in background tissue.

19

Cytotoxicity Product Selection Guide

Follow the selection guide below to determine the appropriate Roche Applied Science product for the study of cytotoxicity to meet your needs. If you need additional help, please visit
www.roche-applied-science.com/apoptosis
Start
LDH released from damaged cells

Cytotoxicity Detection KitPLUS (LDH) no using detection by colorimetric ELISA Do your cells proliferate in vitro? yes
LDH released from damaged cells

04 744 926 001 04 744 934 001

Cytotoxicity Detection KitPLUS (LDH)

BrdU incorporation during DNA synthesis

04 744 926 001 04 744 934 001 11 585 045 001

Cellular DNA Fragmentation ELISA cytotoxic substances and you are studying cell-mediated cytotoxicity
Cytotoxicity
LDH released from damaged cells

Cytotoxicity Detection KitPLUS (LDH)

04 744 926 001 04 744 934 001 11 644 807 001 05 015 944 001

Metabolic activity: tetrazolium salt conversion

Cell Proliferation Reagent WST-1

chemiluminescent ELISA

using detection by

no Do your cells proliferate in vitro? yes

colorimetric ELISA

Metabolic activity: tetrazolium salt conversion

Cell Proliferation Reagent WST-1

LDH released from damaged cells

11 644 807 001 05 015 944 001 04 744 926 001 04 744 934 001 11 647 229 001 11 585 045 001

Cytotoxicity Detection KitPLUS (LDH)

BrdU incorporation during DNA synthesis

Cell Proliferation ELISA, BrdU (colorimetric) Cellular DNA Fragmentation ELISA

in cells that proliferate in vitro

BrdU incorporation during DNA synthesis

Cell Proliferation ELISA, BrdU (chemiluminescent)

11 669 915 001

20

Introduction to Cytotoxicity, Cell Proliferation, and Cell Viability

Cytotoxicity is the cell-killing property of synthesized

Metabolic activity
Cellular metabolic activity can be measured by adding tetrazolium salts to cells. Viable cells convert these salts to colored formazan dyes that are measured spectrophotometrically. ATP levels can also be analyzed as an indication of the energy capacity and, therefore, viability of cells.

chemicals, naturally occurring toxins, or immunemediator cells (e.g., cytotoxic T-cells or natural-killer cells). In contrast to necrosis and apoptosis, the term cytotoxicity does not indicate a specific cell-death mechanism.
Cell viability and cell proliferation assays are commonly used to assess the health of cells. In almost all cases, these assays measure vital functions that are characteristic of healthy cells. Cell viability measurements assess the number of healthy cells in a sample and are often useful in determining optimal culture conditions for cell populations (i.e., when non-dividing cells, such as primary cells, are isolated and maintained in culture). Cell proliferation assays measure the rate at which cells are actively dividing in cultures or tissue.

DNA synthesis
Assessment of DNA synthesis as an indicator of cell growth is a frequently used method for evaluating cell proliferation. Assays are performed using a nonradioactive nucleotide analog, 5-bromo-2'deoxy-uridine (BrdU), which is incorporated into the DNA of proliferating cells. The amount of newly synthesized DNA is then measured by detecting the incorporated BrdU using monoclonal antibodies against BrdU in ELISA (Figure 32), cytochemistry, immunohistochemistry, or flow cytometry.

Many assays for the detection and quantification of cytotoxicity and cell proliferation are based on the use of radioactivity. Roche Applied Science has developed nonradioactive alternatives for these assays and provides products that offer high sensitivity, convenience, and much higher levels of safety. Roche Applied Science offers products for the study of cytotoxicity, cell proliferation, and cell viability via the measurement of: Plasma membrane damage Metabolic activity DNA synthesis

Cytotoxicity and Cell Proliferation

Plasma membrane damage

Assays that monitor membrane permeability involve staining damaged (leaky) cells with a dye and counting viable cells that exclude the dye. Alternatively, membrane integrity is assayed by quantifying the release of substances (e.g., lactate dehydrogenase [LDH]) from cells when membrane integrity is lost.
Figure 32: Measurement of DNA synthesis using modified nucleotides [3H]-TdR and BrdU.

21

Cytotoxicity in Cell Populations

HTS

Cytotoxicity Detection KitPLUS (LDH)

Quantify cytotoxicity and cytolysis with an ELISA in 96- or 384-well plates

Use the Cytotoxicity Detection KitPLUS (LDH) to quickly and easily quantify cytotoxicity/cytolysis in many different in vitro cell systems based on the measurement of LDH activity released from damaged cells. When damage to the plasma membrane occurs (e.g., during cell-mediated cytotoxicity), cells release lactate dehydrogenase (LDH) into the cell culture supernatant. LDH is a stable cytoplasmic protein present at nearly constant concentrations in all cells. Offering improved experimental flexibility, the kit provides all the benefits of the Cytotoxicity Detection Kit (LDH) with the additional advantages of a stop solution and fewer handling steps. Perform high-throughput analysis with minimal handling steps no transfer, centrifugation, or prelabeling steps are required.
Cytotoxicity

Figure 33: Linear range of the Cytotoxicity Detection KitPLUS (LDH). The kit was used to determine total LDH activity from different numbers of U-937 cells in whole cell cultures. This figure shows the values after a 5-minute incubation with the substrate mixture from the Cytotoxicity Detection KitPLUS (LDH).

Control assay conditions by using the kits Stop Solution to terminate the color reaction (optional). Increase safety by eliminating the use of
radioactive isotopes (such as [51Cr] release assays).

Obtain accurate assay results that strongly correlate to the number of lysed cells. Detect low cell numbers (<100 cells/well) with excellent linear range and high sensitivity (Figure 33 and 34). Save time by performing the assay in 96- or 384-well plates, enabling simultaneous processing of multiple samples using an ELISA reader. Use the kit in combination with the WST-1 Assay to obtain more information about cell status (Figure 35).
Sample material: supernatant obtained from cells

Figure 34: Sensitivity of the Cytotoxicity Detection KitPLUS (LDH). The kit was used to determine total LDH activity from different numbers of U-937 cells in whole cell cultures. This figure shows the values for cell numbers down to 100 cells per well after 30 minutes of incubation with the substrate.

Figure 35: Use of the Cytotoxicity Detection KitPLUS (LDH) and the Cell Proliferation Reagent WST-1 to study dose response in U-937 cells. Different concentrations of Actinomycin D were added to the cells and cytotoxicity was measured in the culture using both kits.

cultured in 96- or 384-well plates. 22

Cytotoxicity in Cell Populations

Quickly and easily determine cell status by using the Cytotoxicity Detection KitPLUS (LDH) in parallel with the Cell Proliferation Reagent WST-1 to gain a better understanding of the biological processes occurring in your cell culture (Figure 35, previous page). For more information about the Cell Proliferation Reagent WST-1, see page 25.
Product Cytotoxicity Detection KitPLUS (LDH)
HTS

Cat. No. 04 744 926 001 04 744 934 001

Pack Size 1 kit (400 tests) 1 kit (2,000 tests)

= Suitable for high-throughput screening.

Also available: The Cytotoxicity Detection Kit (LDH), for assessing cytotoxicity using a 96-well ELISA format.
Product Cytotoxicity Detection Kit (LDH) Cat. No. 11 644 793 001 Pack Size 2,000 tests Figure 36: Overview of the Cell Proliferation Reagent WST-1 and Cytotoxicity Detection KitPLUS (LDH) assays. Benefit from easier handling by eliminating transfer, centrifugation, washing, and prelabeling steps in both kits.

Cytotoxicity

Cellular DNA Fragmentation ELISA

Detect apoptosis, necrosis, or cell-mediated cytotoxicity in cell populations with an ELISA

Sample material: culture supernatant and cytoplas-

mic fractions (lysates) of cells with DNA metabolically prelabeled with BrdU (e.g., cell lines and other in vitro-proliferating cells).

Study apoptosis, necrosis, or cell-mediated cytotoxicity by using this versatile kit to measure the fragmentation and/or release of BrdU (5-Bromo-2'-deoxyuridine)-labeled DNA fragments. Use this convenient ELISA format (Figure 37) to: Measure the amount of BrdU-labeled DNA
fragments in cell lysates.

Quantify BrdU-labeled DNA released into culture medium through necrosis or cellmediated cytotoxicity. Compare and quantify the effects of apoptosis-inducing agents on a population of cells.

Figure 37: Principle of the Cellular DNA Fragmentation ELISA. Product Cellular DNA Fragmentation ELISA Cat. No. 11 585 045 001 Pack Size 1 kit (500 tests)

23

Cell Proliferation Product Selection Guide

Follow the selection guide below to determine the appropriate Roche Applied Science product for the study of cell proliferation to meet your needs. If you need additional help, please visit
www.roche-applied-science.com/apoptosis
Start

BrdU incorporation during DNA synthesis light microscopy

BrdU Labeling and Detection Kit II (AP)

11 299 964 001

and you want to measure DNA synthesis

using detection by
FACS or fluorescence microscopy BrdU incorporation during DNA synthesis

In Situ Cell Proliferation Kit, FLUOS BrdU Labeling and Detection Kit I (Fluorescein) If you are studying
cell populations in vitro single cells in vivo or in vitro

11 810 740 001 11 296 736 001

Cell Proliferation

DNA synthesis

and you want to measure

metabolic activity of living cells

using detection by colorimetric ELISA

Metabolic activity: tetrazolium salt conversion

Cell Proliferation Reagent WST-1

11 644 807 001 05 015 944 001

BrdU incorporation during DNA synthesis colorimetric ELISA

Cell Proliferation ELISA, BrdU (colorimetric)

11 647 229 001

using detection by
chemiluminescent ELISA

BrdU incorporation during DNA synthesis

Cell Proliferation ELISA, BrdU (chemiluminescent)

11 669 915 001

24

Metabolic Activity

Assays for metabolic activity using tetrazolium salts

Quantify cell viability, proliferation, or cytotoxicity by measuring metabolic activity in cell populations. Roche Applied Science offers three microplate-based assays that measure metabolic activity via the reduction of tetrazolium salts to formazan salts by viable cells. The most sensitive and convenient of these is the
Cell Proliferation Reagent WST-1.
HTS

Cell Proliferation Reagent WST-1

Measure metabolic activity with a one-step ELISA using a ready-to-use reagent
Figure 38: Use of WST-1 to measure proliferation of CTLL-2 cells in response to human interleukin-2 (hIL-2).

Easily and accurately measure viability, cell proliferation (Figure 38), or cytotoxicity (Figure 39) with the nontoxic, nonradioactive Cell Proliferation Reagent WST-1. The convenient one-step assay is based on the cleavage of the tetrazolium salt WST-1 by viable cells, producing a soluble formazan salt. Simply add the reagent to your cells, incubate, and measure your results without any further handling steps. Accurately measure cell proliferation assay absorbance strongly correlates to the cell number. Detect low cell numbers with higher sensitivity than with MTT and XTT (Figure 40). Save time with a one-step assay and a ready-to-use reagent; no washing steps or additional reagents are required. Normalize cell numbers by using WST-1 in combination with reporter gene assays. Use the kit in combination with the LDH Assay to obtain more information (Figure 35 on page 22).
Sample material: adherent and suspension cells cultured in

Figure 39: Use of WST-1 to determine the cytotoxic activity of human tumor necrosis factor- (TNF-) on WEHI-164 cells.

Cell Proliferation

96-well microplates.
Figure 40: Achieve 5-fold higher sensitivity with WST-1 than with MTT or XTT. P815 cells were preincubated at various concentrations for 20 hours before MTT (), XTT () or Cell Proliferation Reagent WST-1 () was added. After a 4-hour substrate reaction, the absorbance was determined at the respective wavelength with an ELISA plate reader. Product Cell Proliferation Reagent WST-1 Cat. No. 05 015 944 001 11 644 807 001 Pack Size 8 ml (800 tests) 25 ml (2500 tests) Result: The Cell Proliferation Reagent WST-1 has a 5-fold higher sensitivity than the Cell Proliferation Kit I (MTT) and Cell Proliferation Kit II (XTT).

HTS

= Suitable for high-throughput screening.

25

Metabolic Activity

Cell Proliferation Kits (MTT and XTT)

Measure metabolic activity based on tetrazolium salt reduction

In addition to the newer Cell Proliferation Reagent WST-1 (page 25), Roche offers the first-generation Cell Proliferation Kit I (MTT) (Figure 41) and second-generation Kit II (XTT) (Figure 42). MTT was the first tetrazolium salt described and produces a water-insoluble formazan that is solubilized by adding the supplied solubilization solution. In contrast, XTT forms a soluble formazan that can be measured directly without an additional solubilization step. Use these colorimetric assays to: Rapidly measure cell proliferation in response to growth factors, cytokines, mitogens, and nutrients. Conserve resources no washing steps or additional reagents are required. Analyze cytotoxic and cytostatic compounds, such as anti-cancer drugs and other pharmaceutical compounds. Assess growth-inhibitory antibodies and physiological
mediators.
Figure 41: Use of the Cell Proliferation Kit I (MTT) to measure human Interleukin 6 (hIL6) activity on the mouse hybridoma cell line 7TD1. Cells (2 x 103/well) were incubated in the presence of various amounts of hIL-6. After 4 days of incubation, cell proliferation was analyzed by Cell Proliferation Kit I (MTT).

Save time perform the entire assay in one microplate.

Sample material: adherent and suspension cells cultured in

Cell Proliferation

96-well microplates.
Product Cell Proliferation Kit I (MTT) Cell Proliferation Kit II (XTT) Cat. No. 11 465 007 001 11 465 015 001 Pack Size 1 kit (2,500 tests) 1 kit (2,500 tests) Figure 42: Use of the Cell Proliferation Kit II (XTT) to measure human tumor necrosis factor (hTNF-) activity on the mouse fibrosarcoma cell line WEHI-164. After preincubation of the cells (1 x 106/ml) with Actinomycin C (1 g/ml) for 3 hours, cells (5 x 104/well) were incubated in the presence of Actinomycin C and various amounts of TNF- for 24 hours. The cellular response was analyzed by Cell Proliferation Kit II (XTT).

26

ATP Bioluminescence Assay Kits

Assess cell proliferation by measuring ATP levels

Living cells require a continual input of free energy in the form of ATP. Therefore, cell proliferation can be measured by increased levels of ATP. Roche offers two kits for the highly sensitive and quantitative detection of ATP by luciferase-driven bioluminescence. Measure cell proliferation using a well-established technique determination of ATP using bioluminescence. Choose from two formats to meet your application needs. Detect extremely low concentrations of ATP. Study adherent and suspension cells cultured in 96well microplates.
Figure 43: Sensitivity range of the ATP Bioluminescence Assay Kit HS II and the ATP Bioluminescence Assay Kit CLS II.

ATP Bioluminescence Assay Kit HS II

Use the ATP Bioluminescence Assay Kit HS II when you need the highest sensitivity. This kit has a 10-fold higher sensitivity than that of the ATP Bioluminescence Assay Kit CLS II (Figure 43), and is particularly recommended for determinations in the high sensitivity range. An efficient cell-lysis reagent is also provided.
Sample material: eukaryotic and prokaryotic cells.
Product ATP Bioluminescence Assay Kit HS II Cat. No. 11 699 709 001 Pack Size 1 kit (1,000 assays [microplate] 500 assays [tube]) Figure 44: Kinetics of light generation of the ATP Bioluminescence Assay Kit HS II and the ATP Bioluminescence Assay Kit CLS II. ATP (10 pmol) in a volume of 50 l was assayed with 50 l luciferase reagent in a black microplate.
Cell Proliferation

ATP Bioluminescence Assay Kit CLS II

Use the ATP Bioluminescence Assay Kit CLS II when you need high reproducibility due to constant signal generation (e.g., for kinetic studies of enzymes, for metabolic studies, or if coupled enzymatic assays are applied) (Figure 44).
Sample material: eukaryotic and prokaryotic cells.
Product ATP Bioluminescence Assay Kit CLS II Cat. No. 11 699 695 001 Pack Size 1 kit (1,600 assays [microplate] 800 assays [tube])

27

DNA Synthesis in Cell Populations

Detection of BrdU Incorporation

The Cell Proliferation ELISA, BrdU (colorimetric or chemiluminescent) is based on the detection of 5'-Bromo-2'-deoxyuridine (BrdU), a nonradioactive analog incorporated into the DNA of proliferating cells in place of thymidine. Use these second-generation, improved kits for measuring DNA synthesis to: Sensitively detect and quantify cell proliferation induced by growth factors and cytokines. Determine the inhibitory or stimulatory effects of various compounds on cell proliferation.
Sample material: adherent and suspension cell culture.
Figure 46: Comparison of the Cell Proliferation ELISA, BrdU (colorimetric) and the radioactive thymidine ([3H]-TdR) incorporation assay for measuring stimulation of various concentrations of mitogen. Phytohemagglutinin (PHA)stimulated human peripheral blood lymphocytes (PBLs) were labeled for 4 hours with BrdU () or [3H]-TdR (). Result: The Cell Proliferation ELISA, BrdU (colorimetric) () detects mitogen-stimulation with a sensitivity greater than the [3H]-TdR assay ().

Cell Proliferation ELISA, BrdU (colorimetric)

Quantify proliferating cells with a colorimetric ELISA reader

Use the Cell Proliferation ELISA, BrdU (colorimetric) to detect proliferating cells with an ELISA reader. The developed color and resulting absorbance values directly correlate to the amount of DNA synthesis and, therefore, to the number of proliferating cells in the respective microcultures.

Cell Proliferation

Figure 45: Principle of the Cell Proliferation ELISA, BrdU (colorimetric). Product Cell Proliferation ELISA, BrdU (colorimetric) Cat. No. 11 647 229 001 Pack Size 1 kit (1,000 tests)

Figure 47: Comparison of the sensitivity of the Cell Proliferation ELISA, BrdU (colorimetric) and the radioactive thymidine ([3H]-TdR) incorporation assay for measuring proliferation in various concentrations of cells. L929 cells at different concentrations were labeled for 4 hours with BrdU () or [3H]-TdR (). Result: The Cell Proliferation ELISA, BrdU (colorimetric) () measures proliferation with a sensitivity greater than the [3H]-TdR assay ().

28

Benefit from easy-to-use colorimetric or chemiluminescence ELISAs

Obtain results that strongly correlate to the number of proliferating cells (low mean deviation). Avoid the use of hazardous radioactive isotopes by using a nonradioactive method that is at least as sensitive as the [3H]-thymidine incorporation assay, with measurement over a large logarithmic range (Figures 46 and 47). Fix and denature cells in a single step using the kits supplied Fix Denat reagent, then perform only one washing and two incubation steps. Benefit from the convenience of stable and optimized reagents; perform the entire assay in one microplate (Figures 45 and 48). Preserve cell morphology with mild fixation and DNA denaturation methods.

Cell Proliferation ELISA, BrdU (chemiluminescent)

Quantify cell proliferation with a luminescence ELISA reader

Use the Cell Proliferation ELISA, BrdU (chemiluminescent) to detect proliferating cells with a luminescence ELISA reader. The relative light units/second (rlu/s) directly correlate to the amount of DNA synthesis and thereby to the number of proliferating cells in the respective microcultures (Figure 49).

Figure 48: Principle of the Cell Proliferation ELISA, BrdU (chemiluminescent). Product Cell Proliferation ELISA, BrdU (chemiluminescent) Cat. No. 11 669 915 001 Pack Size 1 kit (1,000 tests)

Figure 49: Comparison of the Cell Proliferation ELISA, BrdU (chemiluminescent) and the radioactive [3H]-thymidine incorporation assay for measuring the proliferation of mitogenactivated human peripheral blood lymphocytes (PBLs). PBLs were isolated and cultured in microplates for 48 hours. Subsequently, BrdU [A] or [3H]-thymidine [B] was added and the cells were reincubated for an additional 2 hours (), 4 hours (), 8 hours (), or 24 hours (). BrdU incorporation was determined using the Cell Proliferation ELISA, BrdU (chemiluminescent); the [3H]-thymidine incorporation assay was performed using a standard protocol. Results: The data obtained with the Cell Proliferation ELISA, BrdU (chemiluminescent) strongly correlates to that obtained with the [3H]thymidine incorporation assay.

Cell Proliferation

29

DNA Synthesis in Cell Populations

BrdU Labeling and Detection Kit III (POD)

Measure cell proliferation using a colorimetric ELISA

Measure proliferation of cell populations by the quantitative determination of BrdU (5'-Bromo-2'-deoxy-uridine) incorporated into newly synthesized cellular DNA, using a 96-well ELISA format (Figure 50). Compared to radioactive isotope techniques, the BrdU Labeling and Detection Kit III offers many advantages. Improve safety by avoiding the use of radioactive isotopes. Obtain accurate data results strongly correlate to those obtained with the [3H]-thymidine method (Figures 51 and 52). Achieve high sensitivity using a nonradioactive assay as sensitive as [3H]-thymidine (Figures 51 and 52). Save time by using of multiwell ELISA readers to process a large number of samples. Save money with an assay that requires no expensive equipment or additional reagents, such as scintillation fluid.
Sample material: adherent or suspension cells cultured in
Figure 51: Use of the BrdU Labeling and Detection Kit III () or the [3H]-thymidine method () to measure proliferation of AKR-2B cells (mouse fibroblast cell line) in response to recombinant human epidermal growth factor (hEGF).

96-well microplates.
Cell Proliferation

Figure 52: Use of the BrdU Labeling and Detection Kit III () or the [3H]-thymidine method() to measure proliferation of 7TD1 cells (mouse-mouse hybridoma) in response to recombinant mouse interleukin-6 (mIL-6). Results: Figures 51 and 52 illustrate the equivalent sensitivity of the BrdU and [3H]-thymidine methods in measuring proliferation, as shown in hEGF and mIL-6 stimulation assays. Figure 50: Principle of the BrdU Labeling and Detection Kit III (POD). Product 5-Bromo-2-deoxy-uridine Labeling and Detection Kit III Cat. No. 11 444 611 001 Pack Size 1 kit (1,000 tests)

30

DNA Synthesis in Individual Cells

BrdU Labeling and Detection Kits I (Fluorescein) and II (AP)

Analyze cell proliferation using an immunostaining assay for fluorescence (Kit I) or light (Kit II) microscopy

BrdU Labeling and Detection Kits I and II use

nucleases to denature DNA, allowing binding of the antibody to the BrdU without damaging cell integrity. Cell morphology, including surface and cytoplasmic markers, is preserved, enabling you to: Perform simultaneous detection of other markers (double staining). Label cells in vitro and in vivo. Measure cell proliferation without using
hazardous and inconvenient radioisotopes

Study cell proliferation on a single-cell level by detection of the nonradioactive BrdU (5'-Bromo2'-deoxy-uridine) incorporated into cellular DNA during DNA synthesis. Easily detect cells that have incorporated BrdU by using the kits supplied AntiBrdU monoclonal antibody and an enzyme- or fluorochrome-conjugated secondary antibody. Use the BrdU Labeling and Detection Kit I for immunofluorescence (Figure 54) and Kit II for light microscopy detection (immunocyto/histochemistry) of BrdU incorporated into cellular DNA.

([3H]-thymidine).
Sample material: adherent or suspension cells, fro-

zen or paraffin-embedded tissue sections (after in vivo labeling with BrdU).

Figure 53: Principle of the BrdU Labeling and Detection Kit I (Fluorescein). Figure 54: Fluorescence microscopy-based detection of cells undergoing DNA synthesis. BrdU incorporation was detected using the BrdU Labeling and Detection Kit I. Bright green fluorescence clearly indicates proliferating cells. Product 5-Bromo-2-deoxyuridine Labeling and Detection Kit I Cat. No. 11 296 736 001 Pack Size 1 kit (100 tests)

Figure 55: Principle of the BrdU Labeling and Detection Kit II (AP). Product 5-Bromo-2-deoxyuridine Labeling and Detection Kit II Cat. No. 11 299 964 001 Pack Size 1 kit (100 tests)

Cell Proliferation

31

DNA Synthesis in Individual Cells

In Situ Cell Proliferation Kit, FLUOS

Study cell proliferation using flow cytometry or fluorescence microscopy

Study cell proliferation in many different in vitro and in vivo cell systems at the single-cell level (in situ) by direct immunofluorescence staining for flow cytometry or fluorescence microscopy. After incubation of cells with BrdU, or injection of BrdU into an animal, the BrdU incorporated into the cellular DNA of proliferating cells can be detected by the kits mouse monoclonal antibody directed against BrdU, conjugated to FLUOS (Figures 57 and 58). Measure cell proliferation at the single-cell level, with a safe, easy-to-perform, highly specific assay. Avoid the hazards of radioactivity by using this nonradioactive alternative to tissue autoradiography. Achieve high specificity no cross-reactivity with endogenous immunoglobulins. Save time the kits direct antibody conjugate eliminates the need for a secondary detection system. Follow a standard immunohistochemistry
Cell Proliferation

Figure 57: In vitro labeling and analysis of proliferating HeLa cells with the In Situ Cell Proliferation Kit, FLUOS. HeLa cells in culture were labeled with BrdU, and the BrdUlabeled DNA was detected with anti-BrdU-fluorescein. The labeled cell preparation was analyzed under a light microscope (A) or a fluorescence microscope (B). Result: Proliferating cells (bright green nuclei) within the HeLa preparation are clearly visible under the fluorescence microscope. A

protocol.

Assess cell proliferation using FACS analysis or fluorescence microscopy.

Sample material: cultured or freshly isolated cells,

paraffin-embedded tissue explants or sections.

Figure 58: In vivo labeling and analysis of dorsal, hyperproliferative epidermis tissue from mouse with the In Situ Cell Proliferation Kit, FLUOS. Paraffin-embedded sections of BrdU-labeled mouse tissue were prepared according to standard methods, then digested with trypsin. DNA was partially denatured with HCl and detected with anti-BrdU-fluorescein. Sections were analyzed by differential interference microscopy (A) or epifluorescence microscopy (B). (Data provided by S. Kaiser and M. Blessing, I. Med. Klinik der Universitt Mainz, Germany.) Result: Proliferating cells (green spots) are clearly visible throughout the tissue under epifluorescence microscopy.

Product In Situ Cell Proliferation Kit, FLUOS Figure 56: Principle of the In Situ Cell Proliferation Kit, FLUOS.

Cat. No. 11 810 740 001

Pack Size 1 kit (1,000 tests)

32

Anti-Bromodeoxyuridine Antibodies
Study cell proliferation by detecting BrdU incorporation using antibodies

Anti-Bromodeoxyuridine-Fluorescein formalin grade

For the qualitative measurement of cell proliferation (BrdU incorporation) on a single-cell level; use in cryosections, paraffin sections, flow cytometry, and immunohistochemistry.
Product Anti-Bromodeoxyuridine-Fluorescein formalin grade Cat. No. 11 202 693 001 Pack Size 50 g (250 tests*)

Roche Applied Science offers antibodies for the measurement of cell proliferation via detection of BrdU that is incorporated into cellular DNA. Rely on our high-quality monoclonal antibodies for your cell proliferation studies. Select from several preparations to best meet your needs. Achieve high specificity the antibodies demonstrate no cross-reactivity with other cellular components. Detect BrdU-labeled DNA in proliferating individual cells.
Sample material: cultured or freshly isolated cells,

Anti-Bromodeoxyuridine-Peroxidase, Fab fragments formalin grade

For the qualitative measurement of cell proliferation (BrdU incorporation) on a single-cell level in cryosections, paraffin sections, and immunohistochemistry; use for the quantitative measurement of proliferation in cell populations by ELISA.
Product Cat. No. Pack Size 15 U **

tissue explants or sections.

Anti-Bromodeoxyuridine formalin grade

For the qualitative measurement of cell proliferation (BrdU incorporation) on a single-cell level; use in cryosections, paraffin sections, flow cytometry, and immunohistochemistry. The monoclonal Anti-Bromodeoxyuridine antibody can be used for identifying proliferating cells in blood, tissues, and tumors, as well as for determining plasma cell labeling indices.
Product Anti-Bromodeoxyuridine formalin grade Cat. No. 11 170 376 001 Pack Size 50 g (250 tests*)

11 585 860 001 Anti-Bromodeoxyuridine-Peroxidase, Fab fragments formalin grade * Flow cytometry ** 0.2 U/ml (ELISA), 1.5 U/ml (Immunochemistry)

Cell Proliferation

33

Products for the Study of Apoptosis, Cytotoxicity, and Cell Proliferation

Product Products for Measuring ApoptosisDNA Fragmentation Apoptotic DNA Ladder Kit Cell Death Detection ELISAPLUS HTS Cell Death Detection ELISAPLUS, 10x Cell Death Detection ELISA In Situ Cell Death Detection Kit, AP In Situ Cell Death Detection Kit, Fluorescein In Situ Cell Death Detection Kit, POD In Situ Cell Death Detection Kit, TMR red TUNEL Enzyme TUNEL Label Mix TUNEL AP TUNEL POD TUNEL Dilution Buffer Products for Measuring ApoptosisMembrane Alterations Annexin-V-FLUOS Annexin-V-FLUOS Staining Kit Annexin-V-Alexa 568 Annexin-V-Biotin Products for Measuring ApoptosisCaspase Activity Anti-Poly (ADP-Ribose) Polymerase (Anti-PARP) Caspase 3 Activity Assay Homogeneous Caspases Assay, uorimetric TS
H

Cat. No. 11 835 246 001 11 774 425 001 11 920 685 001 11 544 675 001 11 684 809 910 11 684 795 910 11 684 817 910 12 156 792 910 11 767 305 001 11 767 291 910 11 772 457 001 11 772 465 001 11 966 006 001 11 828 681 001 11 858 777 001 11 988 549 001 03 703 126 001 11 828 690 001 11 835 238 001 12 012 952 001 03 005 372 001 12 236 869 001 12 140 322 001 12 140 349 001 12 156 857 001 04 744 926 001 04 744 934 001 11 644 793 001 11 585 045 001 11 296 736 001 11 299 964 001 11 444 611 001 11 647 229 001 11 669 915 001 11 810 740 001 11 465 007 001 11 465 015 001 05 015 944 001 11 644 807 001

Pack Size 1 kit (20 tests) 1 kit (96 tests) 1 kit (10 x 96 tests) 1 kit (96 tests) 1 kit (50 tests) 1 kit (50 tests) 1 kit (50 tests) 1 kit (50 tests) 2 x 50 l (20 tests) 3 x 550 l (30 tests) 3.5 ml (70 tests) 3.5 ml (70 tests) 2 x 10 ml 250 tests 1 kit (50 tests) 1 kit (250 tests) 250 tests 250 tests 100 l 1 kit (96 tests) 100 to 400 tests 1,000 to 4,000 tests 50 tests 250 tests 250 tests 1 kit (400 tests) 1 kit (2,000 tests) 1 kit (2,000 tests) 1 kit (500 tests) 1 kit (100 tests) 1 kit (100 tests) 1 kit (1,000 tests) 1 kit (1,000 tests) 1 kit (1,000 tests) 1 kit (100 tests) 1 kit (2,500 tests) 1 kit (2,500 tests) 8 ml (800 tests) 25 ml (2,500 tests)

M30 CytoDEATH* M30 CytoDEATH, Fluorescein* Products for Measuring Cytotoxicity Cytotoxicity Detection KitPLUS (LDH)

HTS

Ordering Information

Cytotoxicity Detection Kit (LDH) Cellular DNA Fragmentation ELISA Products for Measuring Cell Proliferation 5-Bromo-2-deoxy-uridine Labeling and Detection Kit I 5-Bromo-2-deoxy-uridine Labeling and Detection Kit II 5-Bromo-2-deoxy-uridine Labeling and Detection Kit III Cell Proliferation ELISA, BrdU (colorimetric) Cell Proliferation ELISA, BrdU (chemiluminescent) In Situ Cell Proliferation Kit, FLUOS Cell Proliferation Kit I (MTT) Cell Proliferation Kit II (XTT) Cell Proliferation Reagent WST-1 S
HT

HTS

= Suitable for high-throughput screening.

*The M30 antibody is made under a license agreement from Peviva AB, Sweden. US Patent No. 6,296,850; 6,706,488 and 6,716,968.

34

Additional Products for the Study of Cells and Proteins

Product Antibiotics Ampicillin BM-Cyclin G-418 Solution Gentamicin Hygromycin B Kanamycin sulfate Mitomycin C Penicillin-Streptomycin (500x) Enzymes for Tissue Dissociation Liberase Blendzyme 1

Cat. No. 10 835 242 001 10 835 269 001 10 799 050 001 04 727 878 001 04 727 894 001 11 059 467 001 10 843 555 001 10 106 801 001 10 107 409 001 11 074 440 001

Pack Size 5g 50 g 37.5 mg (for 2 x 2.5 l medium) 20 ml 100 ml 20 ml (500 x 50 mg/ml) 1 g (20 ml) 5g 2 mg 20 ml (500x) 9 mg 90 mg 6 mg 60 mg 7 mg 70 mg 9 mg 90 mg 500 mg 500 mg 500 mg 500 mg 500 mg 100 mg 500 mg 10 x approx. 2 mg 5x1g 1 kit (25 tests) 1 kit (96 reactions) 10 mg 37.5 mg (for 2 x 2.5 l medium) 100 mg 1 pack 8 ml 20 tablets 0.4 ml 1.0 ml 5 x 1 ml 10 ml 0.4 ml 1 ml Mega-pack 5 x 1 ml 10 ml 1 ml 5 x 1ml

11 988 409 001 11 988 417 001 11 988 425 001 Liberase Blendzyme 2 11 988 433 001 11 814 176 001 Liberase Blendzyme 3 11 814 184 001 11 988 468 001 Liberase Blendzyme 4 11 988 476 001 10 103 586 001 Collagenase A from Clostridium histolyticum, EC 3.4.24.3 11 088 815 001 Collagenase B from Clostridium histolyticum, EC 3.4.24.3 11 088 866 001 Collagenase D from Clostridium histolyticum, EC 3.4.24.3 11 074 059 001 Collagenase H from Clostridium histolyticum, EC 3.4.24.3 11 213 865 001 Collagenase P from Clostridium histolyticum, EC 3.4.24.3 Collagenase/Dispase from Vibrio alginolyticus/Bacillus polymyxa 10 269 638 001 11 097 113 001 04 942 086 001 Dispase I 04 942 078 001 Dispase II Mycoplasma Detection and Elimination 11 296 744 001 Mycoplasma Detection Kit 11 663 925 001 Mycoplasma PCR ELISA 10 236 276 001 DAPI 10 799 050 001 BM-Cyclin Additional Reagents 10 104 159 001 DNase I 11 718 096 001 DAB Substrate, metal enhanced, precipitating 11 681 451 001 NBT/BCIP Stock Solution 11 697 471 001 NBT/BCIP Ready-to-Use Tablets Transfection Reagents 04 709 691 001 FuGENE HD Transfection Reagent 04 709 705 001 04 709 713 001 05 061 369 001 11 815 091 001 FuGENE 6 Transfection Reagent 11 814 443 001 11 988 307 001 05 061 377 001 04 476 093 001 X-tremeGENE siRNA Transfection Reagent 04 476 115 001

Ordering Information

Corresponds to approximately 100 mg crude collagenase (depending on the lot-specific activity in the crude collagenase). 100 mg and 2.5 g packs of these Collagenases are also available. For more information, please visit www.roche-applied-science.com. All Liberase products fall under one or more of the following patents: U.S. Patent No. 5,830,741 or 5,753,495.

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Trademarks LIBERASE and X-TREMEGENE are trademarks of Roche. FUGENE is a registered trademark of Fugent, L.L.C., USA. Dispase is a registered trademark of Godo Shusei Co, Ltd, Tokyo, Japan. Alexa and BOBO are trademarks of Molecular Probes, Inc. Other brands or product names are trademarks of their respective holders. Notice to Purchaser Purchaser represents and warrants that it will use FuGENE Transfection Reagents purely for research purposes. Transfected cells, materials produced, and any data derived from the use of FuGENE Transfection Reagents, may be used only for the internal research of Purchaser whether Purchaser is a for-profit or a not-for-profit organization. Under no circumstances may FuGENE Transfection Reagents be used by Purchaser or any third party for a commercial purpose unless Purchaser has negotiated a license for commercial use with Fugent, LLC (contact information:License@FugentLLC.com). For purposes of the foregoing sentence, commercial purpose shall mean use of FuGENE Transfection Reagents for profit or commercial gain. By using FuGENE Transfection Reagents, Purchaser agrees to be bound by the above terms. If Purchaser wishes not to be bound by these terms, Purchaser agrees to return the FuGENE Transfection Reagents to Roche Diagnostics for a full refund. 2008 Roche Diagnostics GmbH. All rights reserved.

For more information, please visit www.roche-applied-science.com/apoptosis or contact your Roche representative.

Published by Roche Diagnostics GmbH Roche Applied Science 68298 Mannheim Germany 2008 Roche Diagnostics GmbH. All rights reserved. 05173060001 0108