BRDU FACS Method
- Inject mice with 100mg/kg body weight with BrdU (in PBS).
Make sure it’s accurate. Inject again 4 hours later. Kill 1 hour after
final injection. Remove thymus and lymph nodes.
- Cell suspensions and cell counts. Add 3 x 10^6 cells/test.
Controls of cells alone and all single stains do exact same method.
- Surface staining as normal.
- Resuspend in 100uL of PBS/FCS/Az
- Add dropwise to 1ml 1% PFA/0.01% T-20 in eppendorfs. Incubate
o/n at 4 oC.
- Spin tubes down (in eppendorfs). Resuspend pellet in
PBS/2%FCS. Spin twice more.
- Add 500uL of DNase in digestion buffer (2M NaCl, 4.3 mM MgCl
2 ). Incubate in eppendorfs in 37 degree water bath for 30 mins.
- Transfer to v-bottom tubes. Add 1ml PBS/2% FCS. Spin.
Resuspend in PBS/5% FCS (2mls). Spin again.
- Add 40uL of anti-BrdU-FITC (1:2) made up in PBS/5%FCS/0.5%
T-20. Incubate for 30 mins at RT.
- Wash x2 in PBS/FCS/Az. Resuspend in 200uL for FACS.