Cell Counting and Annexin V/ PI Flow Cytometry

Effects of UVB Radiation of Different Intensities on

the Survival and Apoptotic Processes of Human
Fibroblast Skin Cells

Avnika Bali
Catherine Buck
Mohamed Elzarka
Hannah Hales
Jaina Lane
Kelsa Mbah
Bhargav Vemuri

Purpose
To determine the
effects of different
UVB light intensities
on the apoptotic
processes of human
fibroblast skin cells.

Background
Apoptosis is the process of programmed cell death. When a cell undergoes
apoptosis, phospholipid phosphatidylserine (PS), which is usually on the inner
side of the membrane, becomes exposed on the cell surface.
The cell count was conducted to determine the impact of UVB on fibroblasts
over periods of 24 and 48 hours. Flow cytometery was used to determine the
effect of UVB on the cells in one time frame (24 hrs) with regard to apoptotic
processes.

Hypothesis
A higher UVB intensity is directly related to
the number of cells in both early and late
apoptotic stages.
If two different groups of cells which are
irradiated with UVB radiation are allowed
to incubate for 24 and 48 hours
respectively, then the irradiated cells that
have incubated for 48 hours will have fewer
living cells, and the irradiated 48 hr
samples will have more early and late
apoptotic cells.

Plating the Cells

1. Plate cells at 2.5 x 105 cells
into 60 mm dishes. Prepare 9
dishes and add 3 extra dishes
for the flow cytometer internal
controls.
2. 24 to 48 hours prior to the cell counting and flow
cytometry, remove medium and wash cells with sterile
PBS (Phosphate Buffered Saline).
3. Irradiate the cells in PBS with the appropriated doses
of UVB: control (0mJ/ cm2), 75mJ/cm2, 90mJ/ cm2,
and 135mJ/cm2).
4. Remove PBS, add fresh medium and return dishes to
CO2 incubator for 24 to 48 hours.

Irradiation of the Cells

To determine the amount of time that the cell would be irradiated for,
we used a UV meter to measure intensity of the UV light on different
positions 10x. Took the average of the intensies, and divided it by the
amount of UV for each of the cell samples. Since we want the time we
divided whatever we got by 60 minutes, then multiplied the decimal by
60 to get seconds.

Staining Protocol Procedure

1. Remove the medium from
the plates, leaving cells in
the dish intact.
2. Add 1.5 mL of Tyrodes
solution to the dishes and
incubate for 2 minutes at
room temperature.
3. Extract Tyrodes solution
from the dish and discard.
4. Add 300 microliters of
Trypsin (0.25%) to each dish
and incubate at 37oC for 2
minutes.

Staining Protocol Procedure (cont.)

5. Detach the cells by striking
the dishes forcefully to
dislodge any remaining
adhered to the bottom.
6. Neutralize Trypsin with 3 mL
of 4% FBS in PBS.
7. Fill a cell counter dish with
isotonic solution.
8. Extract 1 mL of the cell
culture and put it into the
cell counter vials.
9. Start the cell counting
machine and record the
results.

Staining Protocol Procedure (cont.)

10. Transfer the cell suspension to the 15 mL centrifuge tubes
11. Spin down the cells at 1200 rpm for 5 minutes
12. Carefully remove the supernatant and wash cells with 5 mL
of PBS
13. Spin down the cells at 1200 rpm for 5 minutes
14. Remove the PBS, suspend the cells in 500 mL of [1X] binding
buffer and transfer cells to flow cytometry tubes
15. Add 4 microliters of Annexin V (BD550475) and 15
microliters P.I. (120 microliters)
16. Mix the samples by brief vortexing and keep cells on ice
(because they are alive) and protected from light
17. Immediately take the samples collecting the floating cells
for flow cytometric analysis

Flow Cytometric Analysis

- We used fluorescent labeling molecules: annexin V and PI, which the flow
cytometer detected.
What flow will show us:
- Live cells (No annexin, no PI)
- Early apoptotic (annexin only)
- Late apoptotic (both PI and annexin)
- Necrotic/non-apoptotic death: (PI only)
- Flow cytometry can be used to analyze
multiple parameters, and can detect
chemical characteristics of
thousands of cells per second.
Now that we have a single-cell suspension of stained cells, we are ready for flow!
- After briefly vortexing the cell suspension, we put the sample into the flow
cytometer

PI only;
necrosis

Flow cytometry (A brief review)

-Hydrostatic focusing moves individual
cells through an interrogation point on a
laser light source (what happens if
multiple cells are stuck together?)
- As cells pass through the interrogation
zone, light scatters. Fluorescence emits
in all directions when stained cells pass
through the laser and go into an excited
state
- Detectors are placed around the
interrogation zone, and pick up forward
and side scatter of light.
- Fluorescent light is picked up by
detectors that to pick up a certain
wavelength after the light has been
portioned and filtered

Flow Cytometry Review (cont.)

- Detector picks up changes in light scatter as particles move through the
laser
- Changes are associated with voltage pulses
- As the cytometer takes data, the computer plots
the data for each cell
on a histogram

Cell Count Calculations

Several different data points (2-4) were
taken from the cell counting machine and
then averaged to determine the number of
cells counted in each dish (ex. Control A).
From there, the number of cells counted by
the machine was multiplied by 21 (dilution
factor), and then by 3.3 to find the final
number of cells in each dish.
These final numbers were averaged across
the same group (Average Control).

Cell Count Calculations (cont.)

Cell Count Results

24 hrs

48 hrs

Control

100.000 6.237
(402,733)

100.000 5.079
(530,233)

UVB 90

69.533 16.985
(280,033)

44.251 4.075
(234,633)

UVB 135

44.902 8.819
(180,833)

29.415 4.155
(155,967)

Cell Count Results

Size
Parameter

Raw data
from the
Flow
Cytometer

Control
Population

PI only;
necrosis

live cells (no PI

or annexin)

PI and annexin
are present; late
apoptosis

annexin only;
early apoptosis

events

%parent

%total

All Events

14,422

100%

Cells

11,393

79.0

79.0

Singlets

10,797

94.8

74.9

Q1

0.0

0.0

Q2

0.0

0.0

Q3

10,792

100

74.8

Q4

0.0

0.0

Control- Annexin V
Population

PI only;
necrosis

live cells (no PI

or annexin)

PI and annexin
are present; late
apoptosis

annexin only;
early apoptosis

events

%parent

%total

All Events

13,482

100

Cells

10,267

76.2

76.2

Singlets

9,907

96.5

73.5

Q1

0.0

0.0

Q2

0.0

0.0

Q3

9,189

92.8

68.2

Q4

718

7.2

5.3

Control- Propidium Iodide (PI)

PI only;
necrosis

live cells (no PI

or annexin)

PI and annexin
are present; late
apoptosis

annexin only;
early apoptosis

Population

events

%parent

%total

All Events

13,576

Cells

10,288

75.8

75.8

Singlets

9,884

96.1

72.8

Q1

671

6.8

4.9

Q2

15

0.2

0.1

Q3

9,193

93.0

67.7

Q4

0.1

0.0

100

Control B
Population
PI and annexin are present;
late apoptosis
PI only;
necrosis

live cells (no

PI or annexin)
annexin only;
early apoptosis

events

%parent

%total

All Events

13,482

100

Cells

10,285

75.7

75.7

Singlets

9,914

96.4

73.0

Q1

70

0.7

0.5

Q2

406

4.1

3.0

Q3

9,132

92.1

67.2

Q4

306

3.1

2.3

UV75
PI and annexin are present;
late apoptosis

PI only;
necrosis

live cells (no PI

or annexin)

annexin only;
early apoptosis

Population

events

%parent

%total

All Events

13,670

100

Cells

10,232

74.9

74.9

Singlets

9,929

97.0

72.6

Q1

103

1.0

0.8

Q2

538

5.4

3.9

Q3

8,045

81.0

58.9

Q4

1,243

12.5

9.1

UV90
PI and annexin are present; late
apoptosis
PI only;
necrosis

live cells (no

PI or annexin)
annexin only; early
apoptosis

Population

events

%parent

%total

All Events

13,818

Cells

10,350

74.9

74.9

Singlets

9,883

95.5

71.5

Q1

43

0.4

0.3

Q2

413

4.2

3.0

Q3

6,389

64.6

46.2

Q4

3,038

30.7

22.0

100

UV135
PI and annexin are present; late
apoptosis
PI only;
necrosis

live cells (no

PI or annexin)

annexin only; early

apoptosis

Population

events

%parent

%total

All Events

14,808

100

Cells

10,678

72.1

72.1

Singlets

10,000

93.7

67.5

Q1

72

0.7

0.5

Q2

973

9.7

6.6

Q3

5454

54.5

36.8

Q4

3500

35.0

23.6

Flow Cytometry Calculations

Percentages of the number of cells in each
quadrant were found for each trial (ex.
Control A).
These values were averaged across the
same group for the same quadrant (ex.Q1
Average for Control).
Standard Deviation and Standard Error were
calculated.

Flow Cytometry Calculations

Control

UV75

UV90

UV135

Q3

90.333 1.431

78.667 4.831

64.500 2.858

60.533 3.493

Q4

3.833 0.784

14.167 0.334

29.767 3.896

29.500 3.572

Q2

5.333 0.788

6.367 0.649

5.000 1.007

8.967 0.784

Q1

0.533 0.088

0.733 0.176

0.667 0.176

0.933 0.233

Conclusion
Hypothesis Supported by both Cell Count
and Flow Cytometry Experimentation
Plates that were exposed to higher
intensities of UVB radiation had fewer living
cells accounted for by the cell counter
Plates that were exposed to higher
intensities of radiation also had fewer cells
in Q3 (living), and more in the early (Q4)
and late (Q2) apoptotic stages (as shown by
the data from the flow cytometer).

Conclusion (cont.)
The hypothesis that
the irradiated samples
for the 48 hour plates
would have fewer
living cells and more
apoptotic cells than
the 24 hour plates was
also supported by the
data.

Potential causes of experimental

error
Removing living cells while suctioning solutions
(Tyrodes, Trypsin, etc.)
Leaving Trypsin in the plates for an extended
period of time
Using the same pipette or tube for different
samples
Leaving samples in the centrifuge for too
long/too many revolutions per minute(rpm)
Not vortexing/resuspending/completely
detaching the cells
Adding incorrect amounts of solutions (Tyrodes,
Trypsin, etc.)