FIX & PERM Cell Fixation & Cell Permeabilization Kit

1. For each sample to be analyzed, add the appropriate volume of the conjugated antibody directed to the cell surface marker(s) of interest, and/or the appropriate isotype control(s), to an appropriate 5 mL, 12 x 75 mm tube.
2. Pipette 100 μL of whole blood or adjusted cells (stimulated PBMCs, bone marrow, spleen, thymus), equivalent to 1 x 10⁶ cells, into each tube containing the conjugated antibody or isotype control.
3. Vortex each tube gently to mix, and incubate for 15 minutes in the dark at room temperature.
4. Add 100 μL of Reagent A (Fixation Medium) and incubate for 15 minutes in the dark at room temperature.
5. Wash once with 3 mL wash medium (PBS + 0.1% NaN₃ + 5% FBS).
6. Centrifuge for 5 minutes at 300–350 x g, aspirate supernatant, and vortex to fully resuspend the cell pellet.
7. Add 100 μL of Reagent B (Permeabilization Medium) and the recommended volume of intracellular antibodies or the corresponding isotype control(s).
8. Vortex 1–2 seconds and incubate for 20 minutes in the dark at room temperature.
9. Wash once with 3 mL wash medium (PBS + 0.1% NaN₃ + 5% FBS).
10. Centrifuge for 5 minutes at 300–350 x g and aspirate the supernatant.
11. Resuspend cells in sheath fluid for immediate analysis, or fix in 0.5 mL of 0.1% paraformaldehyde and store at 2–8°C in the dark. Fixed cells should be analyzed within 18 hours.

The methanol modification procedure is recommended for cell cycle antigens such as BrdU, Ki-67, and PCNA when using FITC-conjugated antibodies. It is not recommended when using RPE-conjugated antibodies.

1. For each sample to be analyzed, add 100 μL of adjusted cell volume (equivalent to 1 x 10⁶ cells) to an appropriate 5 mL, 12 x 75 mm tube.
2. Add 100 μL of Reagent A (Fixation Medium) and incubate for 2–3 minutes at room temperature.
3. Add 4 mL of precooled (0–4°C) absolute methanol and vortex.
4. Incubate for an additional 10 minutes at 0–4°C.
5. Centrifuge for 5 minutes at 300–350 x g and wash with wash medium (PBS + 0.1% NaN₃ + 5% FBS).
6. Add 100 μL of Reagent B (Permeabilization Medium) and an appropriate volume of intracellular antibodies or the corresponding isotype control(s).
7. Vortex at low speed for 1–2 seconds and incubate for 30 minutes at room temperature.
8. Wash once with 3 mL wash medium (PBS + 0.1% NaN₃ + 5% FBS).
9. Centrifuge for 5 minutes at 300–350 x g and aspirate the supernatant.
10. Resuspend cells in sheath fluid for immediate analysis, or fix in 0.5 mL of 0.1% paraformaldehyde and store at 2–8°C in the dark. Fixed cells should be analyzed within 18 hours.

VERY IMPORTANT: Blood must be collected into heparinized tubes.

1. Dispense 0.5 mL of heparinized whole blood into each of two 12 x 75 mm snap-cap tubes.
2. Add 0.5 mL of RPMl 1640 (without additives) to each tube to bring the volume to 1 mL.
3. To the first tube, add 10 µg brefeldin A (20 µL of a stock solution of brefeldin A at a concentration of 0.5 mg/mL in 100% EtOH and stored at –20°C). Mix contents of the tube gently. This tube represents the resting cell population.
4. To the second tube, add 10 µg brefeldin A, 25 ng PMA (2.5 µL of a stock solution of PMA at a concentration of 1 mg/mL in DMSO and diluted 1:100 in RPMI 1640 (without additives) and stored at –20°C), and 1 µg ionomycin (1 µL of a stock solution of ionomycin at a concentration of 1 mg/mL in DMSO and stored at –20°C). Mix contents of the tube gently. This tube represents the activated cell population.
5. Incubate for 4 hours at 37°C in a 7.5% humidified CO2 incubator.
6. At the end of the incubation period, mix the cells again and aliquot 100 µL into 12 x 75 mm snap-cap tubes.
7. Add 5 µL of the appropriate antibodies for phenotyping cells (cell-surface staining) (e.g., CD3-TRI-COLOR, CD4-TC, CD8-TC, CD45-TC, or CD69-TC for a three-color staining assay; either FITC or RPE may be appropriate for a two-color assay).
8. Incubate in the dark for 15 minutes at room temperature.
9. Add 100 µL of Reagent A from the FIX & PERM Kit. Mix cells gently and incubate for an additional 15 minutes at room temperature in the dark.
10. Wash twice with wash medium (PBS + 1 % BSA, 0.1 % NaN₃, 1 % FBS). The pellet becomes less cohesive on the second wash, so use caution to avoid losing cells.
11. To the washed cells add 100 µL of Reagent B from the FIX & PERM Kit and 1–10  µL of each anti-cytokine antibody OR isotype control.
12. Incubate for 20 minutes at room temperature.
13. Wash twice with wash medium and resuspend pellet in FACS fix.
14. Analyze by gating on SSC and FL-3 (3- or 4-color assay).