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In Vivo Killing Assay


 
  • Harvest spleen from BALB/c (WT) mouse according to the spleen harvesting protocol. After RBC lysis, wash 1x in complete medium and 2x with HBSS. There are MANY washes throughout this protocol.  To avoid losing a large percentage of cells, I suggest doing every spin at 1200 rpm for 10min.
  • Resuspend at 1x107 cells/mL in plain RPMI (no serum) and split into 2 tubes (one tube for each peptide).  
  • Add AH1 or Bgal peptide (10mg/mL) at a 1:100 dilution (add 10uL per 1 mL of cells).
  • Incubate 2 hrs at room temperature and occasionally mix the cells.  Wash 1x with HBSS.  Resuspend cells again at 1x107 cell/mL in HBSS.
  • From now on, work in the hood with the light off.  Label peptide-loaded cells with a high or low dilution of CFSE.  CFSE stocks (10mM) should be aliquoted and stored at –20oC; pay attention to the expiration date as CFSE can go bad quite quickly. Dilute the stock of CFSE 1:10,000 (1uL in 10mL) for the high dilution in HBSS and mix very well.  Add 1 mL of this dilution to 9 mL of HBSS in a separate tube (1:100,000 dilution) for the low dilution and mix.  
  • Add an equal volume of diluted CFSE to your cells (add 1 mL of diluted CFSE for every 1 mL of cells).  I usually add the high dilution (1:10,000) to the AH1-loaded cells and the low dilution (1:100,000) to the bgal-loaded cells.
  • Incubate at room temperature for EXACTLY 5 minutes (the timing is very important, over-labeling will lead to inconclusive results).  
  • Add an equal volume of FCS to quench the labeling reaction and incubate for 30sec.  
  • Wash 3x with HBSS and resuspend at 2.5x107 cells/mL.  Keep the cells in separate tubes (DO NOT COMBINE).  Keep the cells in separate tubes, in the dark, and on ice until the moment you inject.
  • Once you are set up to I.V. inject in the mouse house (and have some mice warmed up), use a transfer pipet to combine all the cells together in one 15 mL conical tube.  Mix well.  Transfer 1 mL into a 1.5 mL microcentrifuge tube and load the syringe.  The remaining cells can stay in the 15 mL conical tube (on ice) until you’re ready to use them, but mix them before transferring to the 1.5 mL to load the next syringe.
  • Inject 200 uL of the mixed cells (2.5x106 cells from each peptide, or 5x106 cells total) I.V.
  • Harvest spleens or lymph nodes 18-22 hrs later.  I see specific killing in both spleen and lymph nodes at 18 hrs, although waiting until 20 hrs gave  me better results.
  • There is no need to stain your cells for flow, just be sure to have an uninjected (no transfer) animal for settings purposes.  You can save your left-over cells from the injection for flow as well, but the CFSE intensity of all cells will be about a log lower after they have been in the animal.
  • There should be two clearly separate populations of cells, about a log apart.  For flow, I center my 1:100,000 dilution over the 102 intensity and the 1:10,000 dilution over the 103 intensity.  Gate on the CFSE-positive cells and look at them on a histogram.
  • Unvaccinated animals should have an approximately equal number of AH1 and Bgal cells (high- and low-CFSE), and vaccinated animals should have a decrease in the number of AH1-loaded cells.  Collect enough events to have about 1,000 cells in each CFSE positive peak.
  • % specific killing = 1 – % survival; % survival = (# of AH1 targets remaining) / (# of Bgal targets remaining).