Percoll is a polyvinylpyrrolidone-coated silica which is supplied as a sterile colloidal suspension with characteristics which vary slightly from batch to batch. In many cases, monocytes/macrophages are separated on discontinuous (preformed) Percoll gradients. In this case, a relatively large number of density layers is arbitrarily fixed. Consequently, the number of cell subpopulations harvested depends on the number of Percoll solutions which are overlayed in the tubes. An improved cell separation can be obtained by using continuous (or self-generated) density gradient of Percoll. A Percoll solution of a given density is centrifuged and high and low densities are distributed around this given density. The specific cell density of macrophages is influenced by several factors (species, age, sex, strain). For this reason, it is recommended to first determine this specific cell density in an analytical step. This can be done by centrifuging simultaneously a continuous gradient containing coloured beads of well-known cell density together with another gradient of cell preparation to be studied. Subsequently, a preparative step using a discontinuous density gradient allows separation of all the cell subpopulations according to their actual specific density. Protocol 9 describes one procedure for Percoll gradient separation of monocytes.
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