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Typing TG Rag Mice


 
  • Prepare Hank’s Balanced Salt Solution (HBSS) and heparin solution by mixing a bottle of heparin powder into 50 mL of HBSS.  Aliquot 1 mL into each of 50 Eppendorf tubes.  (Do this in the hood).
  • In mouse house:  Warm up mice.  Make small nick at the bottom of the tail where the artery is.  Bleed no more than 5 drops into the Eppendorf tube with the HBSS and heparin.  Stop the bleeding with Kwikstop.
  • Back in the lab:  Spin down the blood cells at 3000 rpm for 5 minutes.  Remove the supernatant. 
  • Add 1 mL ACK lysis buffer.  Let sit at room temperature for 10-15 minutes.  Spin down at 3000 rpm for 5 minutes.  Remove the supernatant.
  • Wash cells by adding 1 mL flow cytometry wash buffer (FCWB).  Spin 3000 rpm for 5 minutes.
  • Remove supernatant.  Transfer cells to 96 well plate, using one well per sample.  Add more FCWB to the top of the wells and spin at 1250 rpm for 5 minutes.
  • Dump out supernatant.  Wash with another 200 uL of FCWB and spin again at 1250 rpm for 5 minutes.
  • Add appropriate antibodies using 10 uL of the appropriate dilution.  When testing to see if the mice are transgenic Rag or not I usually use:
    CD4-FITC
    CD8-Cyc
    Vb8.3-PE
  • Incubate in the refrigerator for 1 hour. 
  • Wash cells 2X with 150 uL FCWB.
  • Transfer to FACS tubes in final volume of 250-300 uL.

Run on the FACScan or FACSCalibur on the 10th floor.