Question

Give any two important methods of cell Seperation.

Answer 1

1. Adherence and Filtration:

Separations of cells using differences in adherence phenomena or filtration properties are among the old­est physical procedures used. Some cells readily ad­here to glass beads, nylon wool, glass wool, and so on and may be separated from nonadhering cells by pass­ing the cell suspension through a hollow glass column packed with these materials.

Success has also been ob­tained by coating glass or plastic beads with antibod­ies, antigens, or haptens so that cells will be differen­tially adsorbed to the beads on the basis of chemical interactions between the plasma membrane and the coating material. Sieves of varying pore diameter can also be used to separate populations of cells on the ba­sis of differences in cell diameter.

2. Conventional and Zonal Centrifugation:

Because of their relatively large size (i.e., in compari­son with organelles and macromolecules) whole cells sediment quite rapidly. Consequently, attempts to fractionate suspensions of cells using centrifugation involve rotation at low rpm (i.e., small RCF) for short periods of time (typically less than 500 g for a few min­utes).

As with subcellular centrifugal fractionations, greatest resolution is obtained using density gradi­ents in which the mixture of cells in the starting zone is separated into subpopulations on the basis of differ­ences in average cell size and/or density.

Most cells be­have like miniature osmometers, so that strict atten­tion must be paid to the selection of gradient solute. Salts are rarely used to prepare density gradients for cell separations because of their deleterious osmotic effects.

Large, impermeable, and biologically inert polymers such as Percoll and Ficoll are the more fre­quent choices. Because they offer the advantage of greatly increased sample size, reograd zonal rotors have been used with great success for separating dif­ferent cell populations.