Studies on a fractionated murine fibrosarcoma: a reproducible method for the cautious and a caution for the unwary

Abstract

A technique is described for the dissociation and fractionation by isopycnic centrifugation (4,000 x g, 60 minutes, 4 degrees C) in linear bovine albumin density gradients (12 ml, pH 5.2 real osmolality 333 mmol/Kg water, 1.030-1.075 g/cm3) of cells ( less than or equal to 3 x 10(7)/gradient) released by a strictly standardised combination of mechanical and enzymatic means from a transplantable methylcholanthrene induced BALB/c fibrosarcoma. Optimal conditions for reproducible localisation of cell bands with maintenance of both satisfactory resolution and satisfactory viable cell recovery ( greater than 80%) were established by means of a series of simultaneous double fractionation experiments. When rebanding was performed under these conditions the density of the median of the cell count of the refractioned cells shifted less than 0.0005 g/cm3. Experiments also showed that close adherence to certain aspects of the tissue dissociation and fractionation protocol was necessary to avoid reduced cell yields and viabilities, density-dependent selective cell lossses, reductions in resolution and shifts in the location of cell bands. Other aspects of the protocol were tolerant to variation without introducting artefacts.