Hair from the skin overlying the left and right dorsal flanks were removed using electrically operated razor. The skin overlying the abdomen was sterilized by wiping with 70% ethanol. Ketamine (1 00 mg/kg) and xylocaine (2%) (20 mg/kg) were mixed and administered intraperitoneally. The mice were returned to the cage and the onset of anesthetic effect was monitored. The mice were considered to be in surgical anesthesia when there was loss of palpebral reflex, righting reflex, and toe pinch reflex. Respiratory rate and heart rate were monitored continuously.

General anesthesia:

Thespleenswereremovedandhomogenisedindividuallythroughanylonnet(80mesh).Forisolationoflymphocytes,thetissuehomogenatewaslayeredonatwo-stepPercolldensitygradientandcentrifugedfor30minat400xg.Thelymphocyteswerecollectedatthe1.040-1.080g/cm3interface,washedtwice(400xg,10min)withrHbss,andresuspendedin2mlrRPMl.Cellswerecountedbytrypanblueexclusioninahaemocytometer(viability>95%)andthenumberoflymphocytesfrombloodandspleenwereadjustedto2x106/ml

Spleen