Protocol Outline

1. Cell Treatment: Prepare single cell suspension and pellet cells by centrifugation. Resuspend cells in 50-100 μL/test of 1× Dulbecco"s PBS containing Ca++/Mg++, and then treat the cells (eg, with a response modifier).

2. Fix and permeabilize the cells with BD Pharmingen™ Transcription Factor Phospho (TFP) Buffer Set (Cat. No. 563239/565575).

3. Wash cells using 1× TFP Perm/Wash Buffer. Pellet cells by centrifugation and aspirate supernatants. Do not leave residual buffer supernatant

4. Incubate cells with BD Phosflow™ Perm Buffer III, pellet cells, and remove Perm Buffer III supernatant.

5. Wash cells with 1× TFP Perm/Wash Buffer and stain cells with fluorescent antibodies. Wash the cells with 1× TFP Perm/Wash Buffer.

6. Resuspend cells in BD Pharmingen™ Stain Buffer (BSA) (Cat. No. 554657) and analyze by flow cytometry.

General Comments

·The entire protocol is performed at 2-8ºC with two exceptions:

oDuring the treatment of fresh cells in 1× DPBS with response modifier

oWhen using Perm Buffer III which has been stored at -20ºC.

·Cool the centrifuge and prepare fluorescent antibody cocktails and other solutions prior to starting the protocol.

·Prepare the 1× TFP Perm/Wash Buffer from the 5× stock TFP Perm/Wash Buffer solution by diluting with filtered, deionized water.

·Also, prepare the 1× TFP Fix/Perm Buffer 10-15 minutes prior to use. Mix 4× TFP Fix/Perm Buffer stock with 0.75× TFP Diluent to make 1× TFP Perm/Wash Buffer (eg, use 10 ml of 4× TFP Fix/Perm Buffer stock and mix with 30 ml of 0.75× TFP Diluent).

·The time interval for staining cells with antibodies can likely be limited to 30 min. However, the original staining protocol was developed using at least 40 minute-incubation times.

·Keep centrifuge at 2-8ºC, and keep centrifuge speeds constant (380 g prior to cellular fixation, 500 g after fixation).

·There is no permeabilization incubation time introduced using the Perm/Wash Buffer. It is not necessary.

·This protocol is not optimized for use with whole blood.

Materials and Equipment

·1× Dulbecco"s PBS (DPBS) containing Ca++/Mg++

·1× Dulbecco"s PBS (DPBS) containing without Ca++/Mg++

·BD Pharmingen™ Transcription Factor Phospho Buffer Set (Cat. No. 563239/565575)

·BD Pharmingen™ Stain Buffer (BSA) (Cat. No. 554657)

·50 or 15 mL sterile polypropylene conical centrifuge tubes

·37ºC incubator or water bath

·Temperature controlled centrifuge capable of 200-600 g and accommodating flow cytometry tubes, 96 deep-well plates, and 15 and 50 mL conical tubes

·Samples: Peripheral blood mononuclear cells (PBMC) 1-2 million cells per test for bulk processing and flow cytometry tube. PBMC 0.75-to-1.0 million cells per test for HTS in deep-well plates and 96-well round bottom plates for HTS on the flow cytometer

·Heat-inactivated FBS with 7.5% DMSO freezing solution

·Flow cytometry tubes or 2.2 mL Storage Plate, MARK II (96 Deep Well; Abgene Cat. No. AB0932) and 96-well tissue culture plates

·Fluorescent antibodies specific for intracellular and cell surface antigens

·Multichannel pipettes that can deliver 20 μL, 200 μL or 1,000 μL volumes

Staining Cells in Tubes

1. Treat cell preparations (human or mouse ) with a response modifier, such as, a cytokine at suboptimal or saturating (eg, 50-100 units per mL) doses of interest, using a 1 mL suspension of 10-20 million cells (10 tests) in 1× DPBS with Ca++/Mg++. Note: Keep this volume of DPBS low, it is important to the intended effect of the 1× TFP Fix/Perm Buffer. Use a 50 mL polypropylene conical centrifuge tube for bulk cell treatment.

2. Incubate 15 min at 37 ºC, exactly, or use another specific treatment condition as desired.

3. Stop cell treatment by adding 1× TFP Fix/Perm Buffer to the cells. Note: Slowly add 10 mL of 1× TFP Fix/Perm Buffer (at 2-8ºC). Cap and invert tubes several times.

4. Incubate for 50 min at 2-8ºC to fix and permeabilize the cells.

5. Add 5 ml of 1× TFP Perm/Wash Buffer.

6. Centrifuge cells at 500 g (15 min at 2-8 ºC).

7. Aspirate supernatants away from pelleted cell samples, wash cells once with 10 mL 1× TFP Perm/Wash Buffer.

8. Centrifuge cells at 500 g (10 min); aspirate all residual supernatant volume away from pelleted cells.

Long Term Storage Option: After step 8, can resuspend cells in 200-500 μL FBS with 7.5% DMSO and freeze at -80ºC in cryopreservation tubes. Thaw by removing cells from freezer, adding equal volumes of cold TFP Perm/Wash Buffer, and thaw in cold water bath. Wash once with TFP Perm/Wash Buffer, and then proceed with the protocol from step 9.

9. Add 1.0 mL Perm Buffer III (or other desired volume if doing in bulk) per 1-2 million cells

Notes: i) Perm Buffer III is taken from a -20ºC freezer. ii) Cell pellets become translucent during this step.

10. Incubate for 20 min on ice to allow exposure of the phosphorylated protein epitopes.

Short Term Storage Option #2: Store overnight at -20ºC. Cover tubes with Parafilm® and protect from moisture. Continue with protocol next day. If choosing to store samples in this manner, remove to 2-8 ºC and allow to warm for 5-10 minutes and proceed to step 11.

11. Centrifuge at 500 g (8 min, at 2-8ºC) and carefully aspirate Perm Buffer III being sure to remove all residual volume.

12. Wash once with 1× TFP Perm/Wash Buffer. Use 10 mL when using 50 mL conical tubes in bulk or 2 mL if using flow cytometry tubes; remove all residual buffer from the cell pellet after centrifugtion. Add 80-90 μL 1× TFP Perm/Wash Buffer per test and resuspend the cell pellets in preparation for staining. Optional: Repeat washing step to be sure to remove all Perm Buffer III. Failing to wash the methanol out well enough prior to staining can lead to destruction of protein fluorochromes and tandem dyes (eg, PerCP, PE, PE-Cy™7).

13. Staining with fluorescent antibodies: At this time it may be necessary or desirable to aliquot the cell samples into flow cytometry tubes for staining with different panels of fluorescent antibodies.

a. Intracellular Staining: Add antibodies specific for intracellular target antigens such as FoxP3, T-bet, phosphorylated Stat proteins, or other intracellular molecules as desired.

Note: It is known that when staining cells in TFP Perm/Wash Buffer, lower concentrations of antibodies are needed for intracellular staining, eg, 0.5× or 0.25× test size (usually 2.5 µL of a 5 µL test size). However, fluorescent antibodies specific for phosphorylated Stat proteins should be used at the stated test size on the vial.

Note: When staining Human FoxP3, antibody clone 236A is recommended for best results.

b. Staining Surface Molecules: Prepare a cocktail of fluorescent antibodies specific for staining cell surface antigens in 1× TFP Perm/Wash Buffer or Stain Buffer (BSA). For example, dilute fluorescent surface antibodies at a 10 µL:1000 µL dilution to make a cocktail of 50 tests at 20 µL per test.

14. Incubate for 40-50 min at 2-8ºC

15. Wash cells once with 2 mL 1× TFP Perm/Wash Buffer, centrifuge cell suspensions at 500 g for 5 min at 2-8ºC, and aspirate supernatants away from the pelleted cells.

16. Resuspend cells in 350 μL Stain Buffer (Cat. No. 554657). Note: do not resuspend cells in 1× TFP Perm/Wash Buffer!

17. Acquire a sufficient number of events by flow cytometry to obtain statistically significant results, eg, ≥ 15,000 CD4+ or ≥ 150 FoxP3+ events, as the case may be. Due to the fixation and permeabilization procedure, forward and side light-scatter signals can be slightly different than those of live cells.

Staining Cells in 96-Well Plates

Use 2.2 mL Storage Plate, MARK II (96 Deep Well; Abgene Cat. No. AB0932) and 96-well tissue culture plates

1. Treat cell preparations (human or mouse) with response modifiers (as above) in 96 deep-well plate using cells at 0.5-1.0 million cells/well in 1× DPBS with Ca++/Mg++. For example: start with 50 μL of cells at 10-20 million cells/mL (0.5-1 million cells/well) and add 20 μL of response modifier (diluted in DPBS + 1 mg/mL BSA), for a final cell + response modifier volume of 70 μL.

2. Incubate 15 min at 37 ºC, exactly, or use another specific treatment condition as desired.

3. Stop cell treatment by adding 1× TFP Fix/Perm Buffer to the cells. Use a 1.2 mL multichannel pipette to add 1.0 mL of cold 1× TFP Fix/Perm Buffer.Mix thoroughly by pipetting up and down several times.

4. Incubate for 50 min at 2-8ºC to fix and permeabilize the cells.

5. Add 350 μL of 1× TFP Perm/Wash Buffer to each well.

6. Centrifuge cells at 500 g for 8 min at 2-8ºC.

7. Aspirate supernatants from samples and wash cells once with 700 μL of 1× TFP Perm/Wash Buffer.

8. Centrifuge cells at 500 g for 8 min and aspirate all residual supernatant volume away from pelleted cells.

Long Term Storage Option: After step 8, resuspend cells in each well with 200-500 μL FBS with 7.5% DMSO and freeze at -80ºC. Thaw by removing cells from freezer, adding 1 mL cold TFP Perm/Wash Buffer, and thawing plate in a cold water bath. Wash cells with TFP Perm/Wash Buffer one additional time, and then proceed with the protocol from step 9.

9. Before adding any buffer to the plate, vortex to resuspend the cells. Add 500 μL of Perm Buffer III per well, and pipette up and down to mix.

Notes: i) Perm Buffer III is taken from -20 ºC freezer prior to use. ii) Cell pellets turn translucent during this step.

10. Incubate for 20 min on ice to allow exposure of the phosphorylated epitopes.

Short Term Storage Option: Store overnight at -20ºC. Cover plate with lid, secure it with Parafilm® and protect from moisture. Continue with protocol next day. If choosing to store samples in this manner, remove plate to 2-8 ºC and allow to warm for 5-10 minutes and proceed to step 11.

11. Centrifuge cells at 500 g for 8 min at 2-8 ºC, carefully aspirate Perm Buffer III supernatant away from pelleted cells.

12. Wash once with 1×TFP Perm/Wash Buffer, 1 mL each per well and centrifuge; remove all residual buffer.

Optional: Repeat washing step to be sure to remove all Perm Buffer III. Failing to wash the methanol out well enough prior to staining can lead to destruction of the protein fluorochrome and tandem dyes (eg, PE, PerCP, PE-Cy™7, etc.). Resuspend cells in 100 μL 1× TFP Perm/Wash Buffer.

13. Staining with fluorescent antibodies:

a. Intracellular Staining: Add antibodies specific for intracellular target antigens such as FoxP3, T-bet, phosphorylated Stat proteins or other intracellular molecules as desired.

Note: It is known that when staining cells in TFP Perm/Wash Buffer, lower concentrations of antibodies are needed for intracellular staining, eg, 0.5× or 0.25× test size (usually 2.5 µL of a 5 µL test size). However, fluorescent antibodies specific for phosphorylated Stat proteins should be used at the stated test size on the vial.

Note: When staining Human FoxP3, antibody clone 236A is recommended for better results

b. Staining Surface Molecules: Prepare a cocktail of fluorescent antibodies specific for staining cell surface antigens in 1× TFP Perm Wash Buffer or Stain Buffer (BSA). For example, dilute fluorescent surface antibodies at a 10 µL:1000 µL dilution to make a cocktail of 50 tests at 20 µL per test. Add this test volume to the tube after adding the intracellular antibodies.

14. Incubate for 40-50 min at 2-8ºC

15. Wash cells once or twice with 200 μL TFP Perm/Wash Buffer, centrifuge at 500 g for 5 min at 2-8ºC; carefully aspirate sample supernatants. Transfer cells to standard, round-bottom 96-well plates for acquisition.

16. Resuspend pelleted cells in 200 μL Stain Buffer (BSA) (Cat. No. 554657). Note: do not resuspend in 1× TFP Perm/Wash Buffer!

17. Acquire a sufficient number of events in order to obtain statistically significant results, eg, ≥ 15,000 CD4+ or ≥ 150 FoxP3+ events, as the case may be.

Antibodies Tested as Compatible for Staining using the BD Pharmingen™ Transcription Factor Phospho Buffer Set

DescriptionCloneReactivityFormats TestedCat. No.

CD3SK7HuPE340662

CD3UCHT1HuPerCP-Cy5.5, 560835, 562427, 560365

BD Horizon™ BV421 and V450

CD4SK3HuBV510, PerCP-Cy5.5562970, 562971, 560650

CD20 HI (FB1)HuAlexa Fluor® 700561171

CD21B-ly4HuAPC559867

CD252A3, MA251HuPE, BD Horizon™ BV421564033, 557138, 341010,

564033, 562442

CD45RAHI100HuPE-Cy™7560675

CD317A2MsAPC-Cy™7, BV421560590, 564008

CD4RM4-5MsPerCP-Cy5.5553050

CD253C7MsBV421564370

Stat1 (pY701)4aHu, MsAlexa Fluor® 647612597

Stat3 (pY705)4/P-STAT3Hu, MsAlexa Fluor® 647557815

Stat4 (pY693)38/p-Stat4Hu, MsAlexa Fluor® 647558137

Stat5 (pY694)47/Stat5(pY694)Hu, MsAlexa Fluor® 647562076, 612599

Stat6 (pY641)23/Stat6, 18/P-Stat6 Hu, MsAlexa Fluor® 647560000, 612601

CD247, CD3 Zeta (pY142)K25-407.69HuAlexa Fluor® 647558489

ZAP-70 (PY319)/Syk (PY352)17A/P-ZAP70HuAlexa Fluor® 647557817

SLP-76 (pY128)H3HuAlexa Fluor® 647560057

AKT (pS473)M8961HuAlexa Fluor® 647560343

LAT (pY171)158-1169HuAlexa Fluor® 647559518

ERK 1/2 (pT202/pY204)20AHuAlexa Fluor® 647561992

Ki-67B56HuAlexa Fluor® 647561126

FOXP3259D/C7HuAlexa Fluor® 488, PE560047, 560046

FOXP3236A/E7HuAlexa Fluor® 488, PE561181, 560852

FOXP3MF23MsAlexa Fluor® 488, PE560407, 561181

T-betO4-46Hu, MsPE, PE-CF594561268, 562467

GATA3L50-823HuPE560074

EZH2 PE11/EZH2HuPE562478

BMI-1P51-311HuPE562528

Pax-51H9HuPE562688

Fixable Viability Stain 450N/AHuBD Horizon™ V450562247

All formats of the antibodies listed were tested and found to work in staining cells using the BD Pharmingen™ Transcription Factor Phospho Buffer Set. This buffer is likely to work with other formats of the same clones as well as other antibodies. However, they have not been tested.