BD Cytoperm™ Permeabilization Buffer Plus is specially formulated for the immunofluorescent staining of incorporated BrdU for flow cytometric analysis and may be found in the BD Pharmingen™ FITC BrdU Flow Kit (Cat. No. 559619 / 557891) or the BD Pharmingen™ APC BrdU Flow Kit (Cat. No. 552598 / 557892). Investigators may find the following abbreviated protocol to be helpful.
1. Immunofluorescent staining of cell surface antigens.
a. Add BrdU-pulsed cells (10^6 cells in 50 µL of staining buffer) to flow cytometry tubes.
b. Add fluorescent antibodies specific for cell-surface markers in 50 µL of staining buffer (eg, BD Pharmingen™ Stain Buffer (FBS) Cat. No. 554656) per tube and mix well.
c. Incubate cells with antibodies for 15 minutes on ice.
d. Wash cells 1x by adding 1 mL of staining buffer per tube, centrifuge (5 min.) at 200 - 300 x g, and discard supernatant.
2. Fix and permeabilize cells with BD Cytofix/Cytoperm Buffer.
a. Resuspend cells with 100 µL of BD Cytofix/Cytoperm Buffer per tube.
b. Incubate cells for 15 - 30 minutes at room temperature or on ice.
c. Wash cells 1x with 1 mL of 1x BD Perm/Wash Buffer, centrifuge as in step 1d and discard supernatant.
3. Incubate cells with BD Cytoperm™ Permeabilization Buffer Plus.
a. Resuspend cells with 100 µL of BD Cytoperm™ Permeabilization Buffer Plus per tube.
b. Incubate cells for 10 minutes on ice.
c. Wash cells 1x by adding 1 mL of 1x BD Perm/Wash Buffer (as in Step 2c).
4. Re-Fixation of cells
a. Resuspend cells with 100 µL of BD Cytofix/Cytoperm Buffer per tube.
b. Incubate cells for 5 minutes at room temperature or on ice.
c. Wash cells 1x by adding 1 mL of 1x BD Perm/Wash Buffer (as in Step 2c).
5. Treatment of cells with DNase to expose incorporated BrdU.
a. Resuspend cells with 100 µL of diluted DNase (diluted to 300 µg/mL in DPBS) per tube, (ie, 30 µg of DNase to each tube).
b. Incubate cells for 1 hour at 37°C.
c. Wash cells 1x by adding 1 mL of 1x BD Perm/Wash Buffer (as in Step 2c).
6. Stain BrdU and intracellular antigens with fluorescent antibodies.
a. Resuspend cells with 50 µL of BD Perm/Wash Buffer containing diluted fluorescent anti-BrdU and/or antibodies specific for intracellular antigens.
b. Incubate cells for 20 minutes at room temperature.
c. Wash cells 1x by adding 1 mL of 1x BD Perm/Wash Buffer (as in Step 2c).
7. Optional - Staining of total DNA for cell cycle analysis.
Note: Proceed to Step 8 if the staining of total DNA levels is not desired.
a. Resuspend cells with 20 µL of the 7-AAD solution (Cat. No. 559925).
8. Resuspension of cells for Flow Cytometric Analysis.
a. Add 1 mL of staining buffer to each tube to resuspend cells.
b. Analyze stained cells with a flow cytometer (run at a rate no greater than 400 events/sec.) and acquire multiparameter data files.
Note: Samples may be stored overnight at 4°C, protected from exposure to light, prior to analysis by flow cytometry.
