A very reliable method to distinguish proliferating from nonproliferating lymphocytes is based on differential staining of RNA and DNA (See the original paper, PNAS, 73: 2881-84, 1976). Nonstimulated lymphocytes have minimal RNA content and upon mitogenic stimulation (G0 to G1 transition) their RNA increases by at least one order of magnitude, reflecting an increase in number or ribosomes. When cells enter S and G2/M phases their DNA also is increased. Differential staining of DNA vs. RNA, thus, allows one to discriminate between Go vs G1vs S vs G2/M cells and detect as few stimulated cells as one per 10E4. They are also detected early, upon entrance G1 phase and prior to initiation of DNA replication. Unfortunately, the companies that sell reagents and flow cytometers discourage the use of this simple, rapid and inexpensive staining (it takes ~ 1 min, the yearly supply of the fluorochromes is below $10.-). perhaps since it is more profitable to sell mAbs that also can detect mitogenically activated cells. It is alleged that one of the dyes that can be used for differential staining of RNA and DNA (acridine orange, AO) produces "irreversible contamination" of flow cytometers. This is, of course, nonsense, and AO is no worse than other strongly fluorescing dyes e.g such as rhodamine 123.. We (and many other other labs that I know) routinelly use AO e.g. on FACScan, followed by immunocytochemical samples, with no problems as long as a rinse is given in between the samples, with a diluted solution of a bleacher (chlorox) and detergent. For differential staining of DNA and RNA one can use either AO or a combination of Hoechst and pyronine Y dyes. The methods are described in Meth. Cell Biol. Vol 41, 402-419, 1994, and in Current Protocols in Cytometry. Zbigniew Darzynkiewicz
This archive was generated by hypermail 2b29 : Sat Mar 10 2001 - 19:31:02 EST