Thanks. You Are Right ! I Was Using Different Definition of Viability - > the Pragmatic

viability

Thanks. You are right ! I was using different definition of viability - > the pragmatic rather than the logical one. I defined cell viability in > terms of survival of tumor cells treated with antitumor drugs - are they > later able to proliferate/ form colonies ?. Since I am working on > antitumor drugs/strategies, I must developed such "tunel vision" in > interpreting live vs dead cells (identifying reproductive cell death with > cell death) > Regards > Zbigniew

Robert, Mitotracker Red CMXRos remains in mitochondria after cell fixation/permeabilization. We do not have experience with Mitotracker Green but Haugland makes note in his Handbook that it does not remain in the cell after fixation/permeabilization. Using non-fixed, live, cells we were able to measure Mitotracker Red, as well as tetramethylrhodamine methyl ester (TMRM, Sigma) fluorescence, each in combination with FLICA (Fig 3 in our recent Cytometry paper), using just 488 nm excitation, in FacsScan. Vermuellen et al., (Exp. Hematol 30, 1107-1114, 2002) also had nice results with TMRM e.g in combination with Annexin V using FaacsScan. We did not check whether TMRM can be fixed. Best wishes and regards Zbigniew

Dear All, Neither MTT nor PI is a reliable assay of cell viability. The PI assay is based on the detection of the loss of plasma membrane capability to exclude this dye. Early apoptotic cells exclude PI. Only necrotic and late apoptotic cells stain with PI. Thus, early apoptotic cells, although for all practical reasons they are dead (certainly reproductively dead), are recognized as live cells by this assay. On the other hand MTT assay measures the "cell redox activity", that is to a large extent mitochondrial but may also be non-mitochondrial (e.g. see Bernas and Dobrucki, Cytometry, 2002;47:236-242). The agents that arrest the cell cycle progression (e.g activating cell cycle checkpoints, inhibitors of DNA polymerase etc) induce unbalanced cell growth. In the absence of DNA replication the cells grow in size, including the increase in mitochondrial mass and activity. Such cells are are moribound - at certain degree of growth unbalance they are irreversibly commited to die. By the MTT assay, however, not only such cells are detectable as live, but with time one sees their increased capacity to reduce MTT. It may appear, therefore that cells in cultures proliferate (this can be seen for up to three days), whereas in fact they are reproductively dead. One has to note that some vendors advertise the MTT kits as "Cell Proliferation Assays", which is quite misleading. The gold standard in viability assays is clonogenicity test. Unfortunately it is cumbersome and time consuming. With each new drug/cell system measured by MTT or other rapid/automatic assays, it is advised to run at least once the clonogenicity test, for comparison.

Zbigniew Darzynkiewicz, M.D., Ph.D.

This is a fabulous way to do viability testing! Once you do this method, you will never do a trypan blue (yech) again. I learned to do this in the Herzenberg laboratory at Stanford, brought it to the VRC--and we've now incorporated it in our clinical trials. Every time we thaw PBMC for doing immune function assays, we assess the viability by fluorescence first (and, in fact, if viability is below a treshhold, I think 60%, we discard the sample). We've even developed an SOP for it. We use a combination of acridine orange and ethidium bromide (not PI)--under a fluorescence scope, "green" is live and "red" is dead--no ifs, ands, or buts--and easily scored by even the most green students with risking a red face. In any case, our procedure is to prepare 3 mg/ml ethidium bromide in absolute ethanol and 5 mg/ml acridine orange in ethanol. Store this stock in a dark vial, refrigerated. To make a working solution, take 1 microliter of each added to 1 milliliter of PBS. This we store at room temp by the fluorescence microscope, and make fresh every few weeks. Please note that AO and EB are considered highly carcinogenic: use gloves and a face mask when preparing the concentrated stock solution, and use gloves when handling the working solution. Dilute cells with an equal volume of the working solution and immediately look on the fluorescence microscope (you can also dilute 1:10 if the cell count is too high). Remember to take this dilution into account when you calculate original numbers. mr (PS, if you don't have EB, you could

I hesitate to disagree with Maryalice, especially given her powerful admonition that whoever says otherwise knows absolutely nothing about PI exclusion... but... "he who hesitates is last". Actually, we found that we can stain with PI, then fix with 0.5% paraformaldehyde, and have the ability to discriminate live/dead for about 2 hours afterward (perhaps as long as 4-6 hours). Waiting overnight, however, is right out--the PI leaks out of dead cells (and, if present in the medium, leaks into live cells). As Mark points out, you should add the PI before the PF, and if you need to wait more than several hours, use EMA (which is considerably less practical for various reasons). We tested this extensively, because of the importance of doing live/dead discrimination, as well as the practicality of fixing cells (for example, from infectious samples). Note that we did not test higher concentrations of paraformaldehyde or other fixatives. mr (PS, with regard to removal of adherent endothelial or tumor cells becoming PI+ : note that a variety of protocols, as asserted already on this list, can transiently permeabilize cells. I would try removing the cells, washing them well in regular medium, waiting 30 minutes, and then adding PI).