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.