A. Solutions and Reagents

  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 distilled water (dH2O). Adjust the pH to 7.4 with HCl and the volume to 1 liter. Store at room temperature.
  2. BrdU (5-Bromo-2′-deoxyuridine) EMD biosciences (Cat. #203806)
  3. Ethanol, anhydrous denatured, histological grade, 100% and 95%
  4. 1.5 M Hydrochloric acid
  5. Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) in 100 mL 1X PBS. Store at 4°C.

B. BrdU Incorporation and Specimen Preparation

  1. Add BrdU to fresh, warm media for a final concentration of 0.03 mg/mL. Set aside.
  2. Collect ~50 million cells in tube by centrifugation and aspirate supernatant.
  3. Add 2 ml of BrdU-containing media to cell pellet, vortex, and incubate at 37°C for 30 minutes.
  4. Pellet cells by centrifugation and aspirate media.
  5. Add 2 ml of cold, 70% ethanol to cell pellet and mix.
  6. Allow cells to fix for 5 minutes at room temperature.
  7. Add 2–3 ml PBS and rinse by centrifugation three times.
  8. Add 1.5 M HCL and incubate for 30 minutes at room temperature.
  9. Add 2–3 ml PBS and rinse by centrifugation two times.
  10. Proceed with Immunostaining section C.

C. Immunostaining

NOTE: Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemacytometer 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 antibody data sheet 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 D1.

D. Optional DNA Stain

  1. Resuspend cells in 0.5 ml of DNA dye (eg. DRAQ5® #4084).
  2. Incubate for at least 5 mins at room temperature.
  3. Analyze cells in DNA stain on flow cytometer.

*Recommended Secondary Antibodies:

Anti-Mouse

posted December 2009

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