Simple and efficient recovery of rare living lymphoid cells from a vast majority of dead cells.

A critical problem associated with establishment of stable transformants of mammalian cells containing drug resistant markers is that many cell types are dependent on cell to cell interaction for growth. Thus, when the transfection efficiency is very low the successfully, but rare transfected drug resistant cells may be inhibited from growing due to low cell density. Moreover, cytotoxicity of the selection drug may reduce survival even further. When dealing with cells growing in monolayers it is sometimes possible to circumvent biological problems of this kind simply by replating the cells to restore the required minimal cell density. However, when experiments involve cells growing in suspension, it can be very difficult indeed to recover the extremely few live cells from the large number of dead ones. It does not help much to reduce the culture volume with the aim of increasing the density of the living cells, because massive amounts of dead or dying cells constitute a considerable source of deleterious lysozymal activity. Nor is it in practice possible to separate minute numbers of live cells from dead cells by centrifugation through a Ficoll gradient without losing the majority of the cells. Alternative methods have involved the use of more elaborate systems with co-transfection of surface markers and subsequent isolation by fluorescence activated cell sorting (6) or magnetic beads (2). To overcome problems of this kind we report here a very simple and inexpensive panning method to separate live cells growing in suspension from an ocean of dead ones. The method is generally applicable and highly specific allowing practically 100% recovery of live cells irrespective of how minute a fraction they constitute of a given culture. The method exploits the specificity of the plant lectin concanavalin A (con A) to a-glucosyl and a-mannosyl residues that are almost universally present on the outer surface of mammalian cell membranes. We observed that living cells, but not dead ones suspended in serum-containing media will adhere strongly to plastic or glass surfaces covalently coated with the lectin. The protocol is as follows: The water-soluble carbodiimide, 1 -cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-ptoluenesulphonate, (WSC) is used to covalently couple con A to the cell culture surface (1). For a 35 mm-diameter petri dish 0.5 ml of WSC (75 mg/ml) (Sigma) and 0.5 ml of con A (15 mg/ml) (Sigma), both dissolved in phosphate buffered saline