The University of Iowa Carver College of Medicine
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)
- 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.
- Adjust cell concentration to 10 x 106/ml, 100µl/sample.
- Add 1ml 0.25% paraformaldehyde while vortexing, incubate in the dark 15 minutes at room temperature.
- Centrifuge 300 x g, 5 minutes, aspirate supernatant.
- Wash x1 in PBS, centrifuge, aspirate supernatant.
- 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.
- Centrifuge; wash x1 in PBS; aspirate supernatant.
- Add 10µl FITC anti bcl-2 or control, mix, incubate 30 minutes in the dark at 4°C.
- Add 2 ml PBS with 2% FCS. Centrifuge, aspirate.
- Add 200µl DNA staining medium; incubate 30 minutes in the dark at 4°C and then keep on ice before running on flow cytometer.
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