STEWART LABORATORY

 

---

 

 

BrdU INCORPORATION PROTOCOL

 

Day 1

 

                                                      1.            Label cells with media containing 100 M BrdU (6 L/mL of 5 mg/mL stock):

 

      ES cells, 30 minutes

 

      3T3 cells, 2.5 hours

 

      Diploid Fibroblasts, 4-6 hours

 

                                                      2.            Trypsinize cells, wash from plate with PBS, spin down (400 x g, 1700 rpm, 5 min in 15 mL tubes no faster!) and re-suspend in 10 mL
70% EtOH

 

                                                      3.            Store at -20C overnight or for several days

 

 

 

 

Day 2

 

 

                                                      1.            Pellet cells and wash once with wash buffer (PBS + 0.5% IFS)

 

                                                      2.            Pellet and re-suspend in 0.5 mL 2M HCl + 0.5% IFS (make fresh!). Incubate at RT for 20 min

 

                                                      3.            Wash cells once with 1 mL wash buffer

 

                                                      4.            Pellet and re-suspend in 0.1M sodium tetraborate (Na2B4O7). Incubate at RT for 2 min

 

                                                      5.            Wash cells x2 with 1 mL wash buffer (set aside a fraction of cells that will be stained with only PIthese will be used in step 10)

 

                                                      6.            Re-suspend cell pellet in anti-BrdU antibody (50L total solution diluted with wash buffer). Incubate for 20 min at 4C

 

 

 

Use BrdU-FITC: Boeringher # 1202693 1:100 dilution (skip to step 9)

 

OR

 

anti-BrdU: Boeringher # 1170376 1:10 dilution

 

 

 

 

                                                      7.            Wash cells once with 1.5 mL wash buffer

 

                                                      8.            Re-suspend cell pellet in anti-mouse FITC antibody (50L total solution diluted with wash buffer, 1:500 dilution). Incubate for 20 min
at 4C

 

                                                      9.            Wash cells x1 with 1.5 mL wash buffer

 

                                                 10.            Re-suspend pellet in 0.3 mL PI (10 g/mL in wash buffer). Incubate for 30 min at RT

 

                                                 11.            Cells are ready for analysis by flow cytometry