Description
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.
Store undiluted at 4°C. Irritating to eyes and skin. Do not breathe vapor. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. Wear suitable protective clothing.
1. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
2. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
3. Cy is a trademark of GE Healthcare.
4. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
