Specific for products:


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, biotinylated, or fluorochrome-conjugated 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. If using a fluorochrome-conjugated primary antibody, resuspend cells in 0.5 ml PBS and analyze on flow cytometer; for unconjugated or biotinylated primary antibodies, proceed to step D9.
  9. Resuspend cells in fluorochrome-conjugated secondary antibody* or fluorochrome-conjugated avidin, diluted in Incubation Buffer at the recommended dilution.
  10. Incubate for 30 minutes at room temperature.
  11. Rinse as before in Incubation Buffer by centrifugation.
  12. 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-Rabbit

Anti-Mouse

Anti-Rat

posted April 2012

Support: 877-678-8324 support@cellsignal.com • Orders: 877-616-2355 orders@cellsignal.com • Web: www.cellsignal.com