Laboratory

Protocol

Babensee Lab.

Intracellular staining for flow cytometry .

First Written: 10/16/14
First Written By: SS
Page: 1 of 3

Last Revised:
Last Revised By:
Version: 1

Materials

Dulbeccos Phosphate Buffered Saline Powder (Invitrogen)
Sodium Azide (Sigma)
Saponin (Sigma # 47036)
Brefeldin-A (Sigma # B6542)
Monesin (Sigma #M5273)
PMA (Sigma #P-8139)
Ionomycin (Calbiochem #407952)
Staining antibodies
100% Ethanol
d.i. water
pH meter (Normalized acid or base to adjust pH)
Filter sterilization equipment(s)

Buffers and Solutions

1x PBS
- Dissolve 1 packet of D-PBS in 1000ml of d.i. water
- pH to 7.2
Note: make 10x PBS 1 packet in 100ml H2O if you dont require 1000ml of PBS for a
single weeks experiments

FACS buffer with BSA/FBS
- 100ml 1x PBS
- 1g (i.e.1%) Bovine Serum Albumin (can use 0.5-1% BSA or 1-5%FBS)
- 0.1 g (i.e. 0.1%) Sodium Azide
- pH to 7.2
- filter sterilize
Note: If using 10x PBS use 10ml of 10x PBS and 90ml H20

10% Saponin solution
- 0.25g of Saponin
- 4.75ml of 1x PBS
Note: If using 10x PBS 475l of 10x PBS and d.i. water to make up 4.75ml
#SSNote For each time freshly weigh out Saponin borrowed from the Prausnitz
stored in 50ml conical tube on shelf
Permeabilization buffer
- 5ml 10% Saponin solution
- 95ml FACS buffer
- pH to 7.2
- filter sterilize

Fixation buffer (4% Paraformaldehyde)
- Warm 80ml of 1x PBS to 60C while stirring

Laboratory Protocol

Babensee Lab.

Intracellular staining for flow cytometry .

First Written: 10/16/14
First Written By: SS
Page: 2 of 3

Last Revised:
Last Revised By:
Version: 1

Add 4g paraformaldehyde to the warm solution

Increase pH by adding normalized base/alkali such as 1N NaOH until the
powder dissolves completely solution should be clear
- Cool down to R.T.
- Add 1x PBS to adjust the total volume to 100ml
- pH to 7.2
- Filter sterilize aliquot and store at -20C for upto 2 months
Note: dissolution should be performed in a fume hood. PFA is noxious when inhaled.

BFA solution
- 5 mg of Brefeldin-A
- 5 ml of 100% Ethanol
- Aliquot and store at 20C.
#SSNote: For pilot/prelims use GolgiPlug solution borrowed from Roy lab BD
GolgiPlug contains 10% brefeldin and 90% DMSO, use 1l per 1ml cell culture media.

Monensin solution
- 5 mg of Monensin
- Make stock solution (2mM)- Dissolve completely in 7.2ml of 100% Ethanol
- Aliquot in 200l and store at 20C.

Species

Cytokines

Transport Inhibitor

Human

IL-1, IL-6, IL-8, TNF-

Monensin

Human

IFN-, IL-2, IL-10, IL-12, MCP-1,

MCP-3, MIG, MIP-1, RANTES

Either Monensin or Brefeldin A

Mouse

IL-6, IL-12, TNF-

Brefeldin A

Mouse

GM-CSF, IL-3, IL-4, IL-5, IL-10

Monensin

Mouse

IFN-, IL-2

Either Monensin or Brefeldin A

Note: Cell stimulation required if activating cells to produce the necessary
intracellular cytokines however, if using ICS for dendritic cells (DCs) , this is not
required as DCs will be stimulated by LPS/TNF/IL-10 etc during final days of
culture. For T-cells used in Mixed lymphocyte reaction, cell stimulation with
PMA/ionomycin can be used as a positive control. ##Also it has been reported
previously (Andersson and Coleclough. 1993. Regulation of CD4 and CD8 expression on
mouse T-cells. J. Immunol. 151: 5123-5134) that the use of PMA and/or Ionomycin for
stimulation of T-cells can result in the decrease or loss of expression of CD4+ and/or
CD8+ expression.

Cell stimulation solution
- PMA stock: 5 mg/ml in DMSO, aliquots of 50 l, store at 80C
- PMA_dilute: 25 l of PMA, 2.5 ml of culture media (final conc. 50
- g/ml)
- Ionomycin stock: 1mM in DMSO

Laboratory Protocol

Babensee Lab.

Intracellular staining for flow cytometry .

First Written: 10/16/14
First Written By: SS
Page: 3 of 3

Last Revised:
Last Revised By:
Version: 1

Cell stim_solution_:
- 20 l of 5mg/ml PMA_dilute
- 13.5 l of 1mM stock of Ionomycin
- 166.5 l of culture medium
Add 10 l of PMA/Ionomycin mix per 1 ml of cells (Final concentrations of PMA
and Ionomycin in media will be 50 ng/ml and 500 ng/ml, respectively).

Procedure

1. Prepare cell culture and plate in 12-well plate if this is to be used for DC
culture, then the cells would already be plated on Day 5 for human PBMCC or
on Day 6 for mouse BMDCC. #also adjust volumes of reagents added in the
subsequent steps to plates with >12 wells to have the same final
concentration
2. Add 10 l of BFA per 1 ml of cells (10g/ml), 8-16 hours prior to antibody
staining or 1l Monensin per 1ml of cells (2M).
Note: If using both Brefeldin and Monensin, use 50% of the volumes mentioned
above of each. See above for when to use Brefeldin or Monensin.
3. Collect and count cells on Day 6 for human or Day 8 for mouse according to
the appropriate protocols (adherent and non-adherent cell fractions) in 15ml
tubes.
4. Spin down at 300xg for 5min and wash with FACs buffer to obtain a
concentration of 106 cells/ml.
5. Stain with antibodies against cell surface markers either in eppendorf tubes
or in a 96-well plate in the dark for 40min at R.T.
6. Add 100l fixation buffer to the cells and incubate for 15-30 min in the dark
at R.T.
Note: In case of having many treatment groups separate them in to different
tubes / wells and add 100l fix buffer to each sample
Note: This is where the ICS procedure begins to diverge from the standard
procedure for analyzing only cell surface markers where you would add 200l
FACS buffer.
7. Spin down at 300xg for 5min and aspirate supernatants
8. Wash with 500l of perm buffer and spin down at 300xg for 5min (Repeat
this step to have a total of 3 washes.)
9. After the third wash, resuspend cell pellets in 200l of perm buffer and stain
with antibodies against intracellular proteins/cytokines.
10. Spins down and resuspend in 200-400l of FACS buffer.

Note: Steps 5-10 MUST be carried out in the dark since the fluorophore-tagged
staining antibodies will easily photo-bleach in presence of light and become
undetectable during flow cytometric analysis. If using 96 well plates for staining, you
can use a black plate to reduce photobleaching or cover your plate entirely with
aluminum foil before placing them in the centrifuge.
Now you are ready to run flow cytometry!!