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 (10^6cells 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 discardsupernatant.

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.