The University of Iowa Carver College of Medicine

Flow Cytometry Facility

Staining DNA and Bcl-2

The following protocol is for simultaneous staining of DNA and intracellular Bcl-2 protein. It uses a combination of paraformaldehyde and methanol for fixation. (protocol courtesy of DAKO)

Reagents

  • Monoclonal mouse anti-human bcl-2 oncoprotein
  • Mouse IgG1/FITC, negative control
  • 70% methanol at -70°C (Note: methanol at -70°C keeps cell aggregation and loss to a minimum)
  • PBS at pH 7.2
  • DNA staining medium
  • 50µg/ml propidium iodide
  • 100 Kunitz units/mL of RNAse A dissolved in PBS
  • 1% paraformaldehyde stock solution: 1 g paraformaldehyde 90 ml ddH2O at 70°C
  • Hold at 70°C for 15-30 minutes in a fume hood. Add 10ml PBS stock solution (10x) and allow to cool. Adjust pH to 7.4. Store at 4°C. Stable for no longer than 3 weeks.
  • 0.25% paraformaldehyde working solution:
  • Mix 1 part of 1% paraformaldehyde with 3 parts of PBS working solution.
  • PBS (10x) 80g NaCl 2g KH2PO4 14.4g na2HPO4, 2H2O 2g KCl
  • Add distilled water to 1 liter. Check pH (pH 7.2). Dilute 1:10 for use.

Staining

  1. Adjust cell concentration to 10 x 106/ml, 100µl/sample.
  2. Add 1ml 0.25% paraformaldehyde while vortexing, incubate in the dark 15 minutes at room temperature.
  3. Centrifuge 300 x g, 5 minutes, aspirate supernatant.
  4. Wash x1 in PBS, centrifuge, aspirate supernatant.
  5. Add 1ml cold 70% methanol. (methanol at -20°C keeps cell aggregation and loss to a minimum) Hold in dark 60 minutes at 4°C.
  6. Centrifuge; wash x1 in PBS; aspirate supernatant.
  7. Add 10µl FITC anti bcl-2 or control, mix, incubate 30 minutes in the dark at 4°C.
  8. Add 2 ml PBS with 2% FCS. Centrifuge, aspirate.
  9. Add 200µl DNA staining medium; incubate 30 minutes in the dark at 4°C and then keep on ice before running on flow cytometer.

References

A Aiello, D Delia, MG Borrello, D Biassoni, R Giardini, E Fontanella, F Pezella, K Pulford, M pierotti and G Della Porta. Flow cytometric detection of the mitochondrial Bcl-2 protein in normal and neoplastic human lymphoid cells. Cytometry 13:502-509, 1992.

Pollice AA, JP McCoy, SE Shackney, CA Smith, J Agarwal, DF Jurhole et al. Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins. Cytometry 13:432-44, 1992