Separation of guinea pig peripheral blood lymphocytes by discontinuous density gradient centrifugation using...

We present a simple method for the recovery of lymphocytes from guinea pig peripheral blood by discontinuous density gradient centrifugation using Ficoll-metrizoate solutions. This technique is formulated specifically for the routine preparation of guinea pig lymphocytes for in vitro cultivation and is capable of recovering 40-60% of available lymphocytes. Whole, heparinized (10-100 U/ml) blood was drawn from strain 2 guinea pigs by cardiac puncture, diluted 1 : 4 with Ca2,Mga free Hanks' balanced salt solution (CMF-HBSS) containing EDTA (5 mM) and gentamicin (50 microgram/ml), and incubated at room temperature for 30-60 min to promote leukocyte disaggregation. Five volumes of diluted blood were layered onto 2 vol of Ficoll-metrizoate adjusted to a density of 1.107 g/ml with sodium metrizoate solution. A band of mononuclear cells (80-90% lymphocytes, 10-20% monocytes, and less than 2% granulocytes) formed at the interface after centrifugation (400 X g, 20-40 min, room temperature). More than 95% of the cells were viable by trypan blue exclusion. Lymphocytes recovered from as little as 3-5 ml whole blood were more sensitive to antigen- or mitogen-activated transformation than leukocyte suspensions obtained by dextran-citrate sedimentation with or without nylon column filtration.