Flow Cytometry

NOTE: Prepare solutions with Milli-Q or equivalently purified water.

1. 1X Phosphate Buffered Saline (PBS): Dissolve 8 g NaCl, 0.2 g KCl, 1.44 g Na₂HPO₄ and 0.24 g KH₂PO₄ in 800 ml dH₂O. Adjust the pH to 7.4 with HCl and the volume to 1 L. Store at room temperature.
2. Absolute Methanol
3. Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) in 100 ml 1X PBS. Store at 4°C.
4. Permeabilization Buffer: 0.5% Triton X-100, 0.2 µg/ml EDTA and 1% BSA in PBS.

A. Isolation of Cells and Fixation

1. Collect cells by centrifugation and aspirate supernatant.
2. Resuspend cells briefly in 100 µl PBS.
3. Add 500 µl of permeabilization buffer. Incubate for 15 minutes on ice.
4. Add 3 ml of ice-cold 100% methanol and mix on and off for 10 minutes.
5. Proceed with staining or store cell at -20°C.

B. Immunostaining

NOTE: Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemocytometer or alternative method.

1. Aliquot 0.5–1x10⁶ cells into each assay tube (by volume).
2. Add 2–3 ml Incubation Buffer to each tube and rinse by centrifugation. Repeat.
3. Resuspend cells in 100 µl Incubation Buffer per assay tube.
4. Block in Incubation Buffer for 10 minutes at room temperature.
5. Add the unconjugated primary antibody at the appropriate dilution to the assay tubes (see individual antibody datasheet for the appropriate dilution).
6. Incubate for 1 hour at room temperature.
7. Rinse as before in Incubation Buffer by centrifugation.
8. Resuspend cells in fluorochrome-conjugated secondary antibody*, diluted in Incubation Buffer at the recommended dilution.
9. Incubate for 30 minutes at room temperature.
10. Rinse as before in Incubation Buffer by centrifugation.
11. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to step C1.

C. Optional DNA Stain

1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
2. Incubate for at least 5 minutes at room temperature.

Analyze cells in DNA stain on flow cytometer.

*Recommended Secondary Antibodies:

Anti-Mouse

• Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 488 Conjugate) #4408
• Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 594 Conjugate) #8890
• Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 647 Conjugate) #4410
• Anti-Mouse IgG (H+L), F(ab')₂ Fragment (PE Conjugate) #8887