The leakage rate of fluorescein (from fluorescein diacetate, #3 in the figure below)) from live cells is really too fast. We normally recommend calcein AM (#1 in the figure below) as the green-fluorescent "viability probe" for both imaging and flow cytometry. BCECF AM is also suitable but the fluorescence of its intracellular product (BCECF) is pH sensitive, whereas that of calcein is not. The fast leakage rate of fluorescein makes it difficult to get reproducible results because the initial intensity of the live cells decreases so fast and also makes the time zero fluorescence difficult to measure Loading and retention characteristics of intracellular marker dyes. Cells of a human lymphoid line (GePa) were loaded with the following cell-permeant acetoxymethyl ester (AM) or acetate derivatives of fluorescein: 1) calcein AM (C-1430, C-3099, C-3100), 2) BCECF AM (B-1150), 3) fluorescein diacetate (FDA, F-1303), 4) carboxyfluorescein diacetate (CFDA) (C-1354) and 5) CellTracker Green CMFDA (5-chloromethylfluorescein diacetate, C-2925, C-7025). Cells were incubated in 4 µM staining solutions in Dulbecco's modified eagle medium containing 10% fetal bovine serum (DMEM+) at 37°C. After incubation for 30 minutes, cell samples were immediately analyzed by flow cytometry to determine the average fluorescence per cell at time zero (0 hours). Retained cell samples were subsequently washed twice by centrifugation, resuspended in DMEM+, maintained at 37°C for 2 hours and then analyzed by flow cytometry. The decrease in the average fluorescence intensity per cell in these samples relative to the time zero samples indicates the extent of intracellular dye leakage during the 2-hour incubation period. [Image] This discrimination is probably best done in combination with ethidium homodimer-1 for dead cells, although propidium iodide is almost as suitable (ethidium homodimer is less likely to be taken up by apoptotic cells, however, than is propidium iodide.) Normalized fluorescence emission spectra of calcein (C-481) and DNA-bound ethidium homodimer-1 (EthD-1, E-1169), both of which can be excited at 488 nm by the argon-ion laser. [Image] "Bergh, Ole" wrote: > > > Fluorescein diacetate will stain live cells green, ethidium bromide > will counterstain red for dead cells. For further details: > > Takasugi, M. An improved fluorochromatic cytotoxic test. > Transplantation 1971 Aug 12(2):148-51 > > Ole J. Bergh, Supervisor > Flow Cytometry Facility > University of Pittsburgh Cancer Institute > 200 Lothrop Street W 1009 Biomedical Science Tower > Pittsburgh, PA 15213 > Phone:412 624 0399 Fax: 412 624 9624 > mailto:berghoj@msx.upmc.edu <mailto:berghoj@msx.upmc.edu> > http://pci.upmc.edu/internet/flowcyto/flow.htm > <http://pci.upmc.edu/internet/flowcyto/flow.htm> > > -----Original Message----- > From: Michal Abel [mailto:abelm@necker.fr] > Sent: Friday, February 01, 2002 3:43 PM > To: Cytometry Mailing List > Subject: staining for viability > > > I've got a question for the list regarding staining for viability. > I am working with freesed/defrost lymphocytes. One of the drawbacks > of this procedure is the number of death cells, which make higher the > background noise. I wonder what kind of marker should I use for > gating only the alive lymphocytes. Logically, I thought, the best > would be necrosis marker like PI. However, I am not sure if I also > need to eliminate the early apoptotic cells or not ? > I would be grateful if someone could help me resolving this problem. > > Thanks, > > -- > Michal Abel, MD, PhD > Laboratory of Clinical Immunology > INSERM U25 > Necker Hospital, 161 Rue de Sèvres. Paris > tel: 33 1 44 49 52 23 > fax: 33 1 44 49 53 74
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