Experimental conditions used to isolate hepatocytes from rats at various ages using a two-stage collagenase perfusion

Rat Age
<1 Week1 Week2 Weeks3 Weeks4 Weeks>5 Weeks
Anesthesia100–300 μl mixture of 10 mg ketamine and 0.8 mg xylazine, quantity sufficient to 1 ml with sterile water40–50 mg/kg ketamine + 5 mg/kg xylazine; i.p.120 mg/kg ketamine + 24 mg/kg xylazine; i.p.
Cannula size (G)2424242424/1616
Ca2+ free buffer (+ collagenase): flow rate, ml/min4–8810–15152030
First-stage perfusion duration (Ca2+ free buffer), min5558810
Collagenase in perfusate, mg/ml0.8–1
Second-stage perfusion duration (plus collagenase), min4-8