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Splenocyte isolation

 
  • Harvest spleen into 5 ml M medium (can be put into a 15 ml conical and brought down to the mouse-house with you).
  • Transfer medium and spleen into a small culture dish.
  • Smash spleen with the top of a sterile 10 ml syringe (the textured end).
  • Pipette through a 40-micron filter into a 15 ml conical tube (if you’re doing a bunch of spleens, you can use these filters at least twice).
  • Bring up to 15 ml with M medium.  Spin down at 1200 rpm for 10 min (this 10 min spin is important for maximum recovery).
  • Decant and resuspend in 2 ml ACK lysis buffer.  Incubate ~3 minutes.
  • Bring volume up to 15 ml with M medium.  Spin down again as before.
  • Decant and resuspend in another 15 ml M medium for the final wash.
  • We usually resuspend in 10 ml and count the splenocytes at a 1:1 dilution with trypan blue.
  • You should recover about 1-2x108 cells per spleen.
      

T depletion using MACS column

  • Degas MACs buffer (1 hour)
  • Spin down splenocytes and resuspend in 1 ml degassed MACS buffer and 20 ul of anti-Thy1.2-bio.  Incubate at 4o for 15 min.
  • Bring up to 15 ml with MACs buffer (degassed), spin 10 minutes at 1200 rpm.
  • Resuspend in 800 ul MACs buffer and 200 ul anti-biotin beads.  Incubate at 40 for 15 minutes.  Bring volume up to 15 ml with MACS.  Spin.
  • Resuspend in 500 ul MACs buffer.
  • Unwrap LS column and place in magnetic holder.  Equilibrate the column by running 3 ml degassed MACs buffer through the column by gravity, collect in a waste 15 ml conical.
  • Slowly add the 500 ul of labeled cells in MACs buffer.
  • Allow the cells to flow through by gravity.
  • Wash the column 3 times with 3-5 ml of MACS buffer (more washing = purer populations).
  • Collect and save the flow-through and column-washes for FACS analysis (negative sample).
  • Remove the column from the magnetic holder, place in a 15 ml conical away from the magnet.
  • Apply 5 ml of MACs buffer to the column, allow about 1 ml to flow through by gravity then flush the rest out by pushing the plunger into the column. 
  • Collect these cells in the 15 ml conical (positive sample). At this point you can spin down the cells and resuspend them in M medium, usually 3 ml.
  • Count at a 1:1 dilution with trypan blue.  Save a small amount for flow analysis. Recovery is usually around 107 cells per spleen.