BrdU (Bu20a) Mouse mAb (PE Conjugate) #50230

BrdU - Flow Cytometry

A. Solutions and Reagents

1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water mix.
2. BrdU (5-Bromo-2'-deoxyuridine)
3. Ethanol, anhydrous denatured, histological grade, 100% or 95%
4. 1.5 M Hydrochloric Acid
5. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4 °C.
6. Recommended Anti-Mouse secondary antibodies: (if needed):
   • Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 488 Conjugate) #4408
   • Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 594 Conjugate) #8890
   • Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 647 Conjugate) #4410
   • Anti-Mouse IgG (H+L), F(ab')₂ Fragment (PE Conjugate) #8887

B. BrdU Incorporation and Specimen Preparation

1. Add BrdU to fresh, warm media for a final concentration of 0.03 mg/mL. Set aside.
2. If using suspension cells, pellet up to 50 million cells by centrifugation in a tube and aspirate supernatant.
3. For suspension cells, add 2 ml of BrdU-containing media to cell pellet, vortex.
4. If using adherent cells, dissociate from culture dish.
5. For adherent cells, add sufficient media to cover cells. Incubate at 37°C for 30 minutes.
6. Pellet cells by centrifugation and aspirate media.
7. Add 2 ml of cold, 70% ethanol to cell pellet and mix.
8. Allow cells to fix for 5 minutes at room temperature.
9. Wash by centrifugation in excess 1X PBS. Discard supernatant. Repeat.
10. Add 1-2 ml 1.5 M HCL and incubate for 30 minutes at room temperature.
11. Wash by centrifugation in excess 1X PBS. Discard supernatant. Repeat.
12. Resuspend cells in desired volume of PBS.
13. Proceed with Immunostaining section C.

C. Immunostaining

NOTE: Count cells using a hemacytometer or alternative method.

1. Aliquot desired number of cells into tubes or wells. (Generally, 5x10⁵ to 1x10⁶ cells per assay.)
2. Pellet cells by centrifugation and discard supernatant.
3. Resuspend cells in 100 µl of diluted antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
4. Incubate for 1 hour at room temperature.
5. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
6. If using a directly conjugated antibody, skip to step 9. For unconjugated antibodies, resuspend cells in 100 µl of diluted fluorochrome-conjugated secondary antibody (prepared in Antibody Dilution Buffer at the recommended dilution).
7. Incubate for 30 minutes at room temperature. Protect from light.
8. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
9. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to step D1.

D. Optional DNA Stain

1. Resuspend cells in 0.5 ml of DNA dye (eg. DRAQ5^® #4084).
2. Incubate for at least 5 min at room temperature.
3. Analyze cells on flow cytometer.