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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.
Sheridan et al. (1977) studied this question.