cell viability can be tested with several vital staining protocols. I always prefere double staining using fluorescein diacetate and propidium iodide. Living cells take up fluorescein diacetate and exclude propidium iodide. Thus, fluorescein diacetate will be cleaved by intracellular lipases yielding green cytoplasmic fluorescence, whereas dead cells will take up propidium iodide yielding red nuclear fluorescence. Cell proliferation can be studied as a function of DNA synthesis by the incorporation of tritiated thymidine, or bromodeoxyuridine following 3H scintillation counting, fluorescence microscopy, or flow cytometry. Whatever your needs are please check the following link:

http://assay.nih.gov/assay/index.php/Section12:Methods_for_Evaluating_Cell_Proliferation

cell viability can be tested with several vital staining protocols. I always prefere double staining using fluorescein diacetate and propidium iodide. Living cells take up fluorescein diacetate and exclude propidium iodide. Thus, fluorescein diacetate will be cleaved by intracellular lipases yielding green cytoplasmic fluorescence, whereas dead cells will take up propidium iodide yielding red nuclear fluorescence. Cell proliferation can be studied as a function of DNA synthesis by the incorporation of tritiated thymidine, or bromodeoxyuridine following 3H scintillation counting, fluorescence microscopy, or flow cytometry. Whatever your needs are please check the following link:

http://assay.nih.gov/assay/index.php/Section12:Methods_for_Evaluating_Cell_Proliferation

Usually, several methods can be adopt. it's base on your microscope, under flurocence or confocal microscope. you can use the PI to stain the dead cell; DAPI for live cell or Combination product :LIVE/DEAD® Cell Viability Assay. Both above methods can not use for living cell observation, Another one is BrdU, but it need to be immunostained with antibody. Under general light microscope, Neutural red can staining, but it hard to identify cell number (sometime we use it to fast screen). Upon my study, we choose the distinct time point :12h, 24h, 2d, 3d, 4d, 5d to evaluate the cell viability and proliferation. you should be note the cell proliferation may be extremely different upon your transfection method. Good luck by Bruce Chen

Very good link George, I would just add another assay - proliferation using CFSE.

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0030912

There is a live/dead stain available in which CFSE enters live cells and stains them green while eosin enters dead cells and stains them red. it used to be marketed by Molecular Probes and I have forgotten the name of the company by whom they have been acquired. For proliferation versus cell death EdU is more expensive than BrdU but is more compatible with a number of immunostains. It is also worth mentioning that simple counting of cells on successive days is both cheap and informative, It should be remembered that a full picture of what goes on in cultures usually requires investigation by at least two complementary techniques.

Terry very nice idea, perhaps you are refering to Invitrogen that acquired molecular probes

I think you are right. it is Invitrogen. We have used the CFSE dilution on myoblasts where it has some limitations due to slow leakage, which does not seem to occur with lymphocytes for instance. it is well worth checking this for any individual cell type.

The live dead imaging kit from Life etchnologies R37601 will be a good one for looking at live cells in green channel and dead cells in red channel.

There are a couple of stains that can be used for counting viable cells. Propidium Iodide (PI) is one of the simplest to use. You count stained cells in comparision to unstained (living) cells with a counting cell (like Neubauer-improved 0,1 mm depth or Fuchs-Rosenthal 0,2 mm depth) to count cells in a specific volume. Alternatively there are various automatic cell counters - e.g. ones with disposable counting cells like ADAM-MC. Pricing of counting cells is €50 and up, ADAM-MC is around €5000 plus consumables of about €0,80 per test.

http://scienceservices.eu/en/adam-mc-automatic-cell-counter.html

There are some very good suggestions here for comparing live/dead cells. Since the question asks for microscopy based methods, I would also suggest comparing the proliferative indices of the shRNA and control groups by immunoflurorescence staining for MIB1/Ki-67.

A very simple method is the Trypan blue assay (for light microskopy only!). Living cells are excluded from staining, while dead or dying cells will be stained blue. You can count them directly. But for further investigations (apoptosis/necrosis) you have to use immunofuorescence with all the nice assays described above (LIVE/DEAD, propidiumiodide, DAPI, annexin V and so on).

Unfortunately, all the mentioned methods will not work reliably with anucleate cells and/or cells that undergo easy artefactual activation. Please, consider this fact when adopting a given method to the cells you are interested in. However, as far as you mention cultured cells used for transfection this should probably not be a problem.

Dear Cesary,

I agree partially, because it's not the case for trypan blue staining as it concern solely a membrane reaction ( so even with anucleated cells) , where death cell cannot re-expel the blue after its entry because of lack of ATP source due to their death (dead cells remain blue).

But a problem with trypan blue is it cytotoxicity; the reason to look at the cells immediatly after staining because if you wait too much, all cells become blue (dye).

Regards

Didier J

besides the live/dead nuclear staining already mentioned, there is the old-fashioned Acridine orange (AO). It has green fluorescence when bound to DNA, and red fluorescence when bound to RNA. Dead cells will have DNA, but not much RNA, since RNA is degraded faster. So dead cells give only green signal, while live cells will have both green and red signals. of course the caveats listed above by Didier, Cezary and apply, and also AO bleaches fairly easily, but it is pletty simple to try. I would have a look at Invitrogen "Cellular Analysis Product Catalog". In chapter 6, it lists a number of reagents for assays for apoptiosis, cell proliferation and for clellular viability

Stan

I love old-fahioned dyes. Does AO work on fixed cells/tissue?

Two reasonably good stains (I say this as a caveat - they can both be subjective) are Trypan Blue and TUNEL assays. The latter will assess DNA fragmentation resulting from apoptotic/necrotic cascades in the cells.

If you want to quantify the rate of proliferation, may be - you can try growth curve assay. Details can be found in "Culture of Animal cells" by R. Ian Freshney. Page 345- .

Acridine orange can be used on both live and fixed cells but in live cells it concentrates in the lysosomes and fluorescesces intensely bright red. This tells you they were alive when you applied the acridine orange but the photoactivation bursts the lysososomes and kills the cells quite rapidly. A quick photograph will give you something to count as an endpoint measurement of viable cell numbers but of course you cannot rescue any of the cells you have actually observed. If you restrict your observation to a small part of the culture, the remaining cells should remain alive until you observe them - although I have never tried this.

EDU (5-ethynyl-2´-deoxyuridine, Invitrogen) is a great alternative to BRDU/ Ki67 measures of proliferation, very reliable and specific in tissues or culture. Although I think they only have fluorescent stains. Best results for quantification by Flow cytometry, microscopy is a little subjective. EDU works for both techniques.

I don´t know what kind of cells you are using but it´s possible to analyze viability with many methods. You may employ acridine orange staining. With these method you can count viable, necrotic and apoptotic cells using a fluorecence microscope. You may also use MTT or ethidium bromide/propidium iodide to analyse cell viability. MTT is also used to analyze proliferation.

Based on the body of the question and not on the title.

Do the transfections and selection. Then plate the same number of untransformed, kockdown and scrable cells in different dishes. wait for a couple of days, then count them via hemocytometer.

Assays like MTT can be confusing as they are mitocondrial activity assays .

The problem with vital stains is that they are usually also cytotoxic and can not be ideally used for cell cultures because this may result in cell death or if even in micro-concentrations may alter the physiologic processes of the cells in culture. These vital stains are therefore more useful in the later part of the evaluation of the cell culture particularly if cell preservation or regeneration of the primary cell culture is a primary concern. These stains are most useful for cell cultures that can be divided to sacrifice some of the cells to evaluate viability. We have done vital staining in the corneal endothelium (which has the least ability to divide) which are highly sensitive to chemicals, osmotic and pH changes which usually results in cell death. What we found useful to monitor the cell culture is through contrast phase microscopy where the cytoplasm, nucleus and nuclear materials can be observed with the use of only light. The cells that are dying can typically show disruption of the cell membrane, swelling of the cell body and nucleus, shrinkage of the nuclear membrane, condensation and fragmentation of the chromatin material. With this technique, a big concern will be cell culture contamination as we have experienced usually observed on the second week onwards. For studies of proliferation or regeneration we have devised standard technique to count and analyze cell morphology by using our cell analyzer/counter used with our specular microscope which can assess the density of the cell culture (originally used to assess corneal endothelial density in vivo or clinically). This is done by getting photograph of the cell culture at a fixed distant and magnification. Cell counts, morphology and density can be expressed as no of cells/mm2 or percentage of both cells that are dead and alive

For vital staining acridine orange and methylene blue were used to verify finally at the end of the observation period or when biomolecules or chemicals are introduced into the cell culture for stimulation of growth, proliferation and regeneration.

Hope this helps

I will simply suggest you to go for I fluorescein diacetate or propidium iodide for cell viability, its best known methods, use flow cytometry if you have huge noog cells....

its very simple method ....

I agree with the majority of colleagues about that fluorescent dyes can be helpful, when determining the ratio dead / live cells. I think is Okay what has been said regarding the flow cytometry. The statement microscopy is a rather subjective affair, however, I would like to talk again. Indeed, there are reliable methods for instance to perform cell counts.

Finally, to quantify cell proliferation, I would also suggest the protein Ki67 (immunocytochemistry). So you can detect very well, for example, the number of cells in the S phase (nuclei marked full).

Just only pointing also to the former thread in MICROSCOPY:: https://www.researchgate.net/post/Cell_viability

Pedro, to clarify my point regarding subjective microscopy. It is more to do with cell numbers, flow cytometry analyses all of a population of cells. numbers are typically in the thousands or tens of thousands (increasing accuracy). Microscopy involves analysis of many less cells and requires the researcher to pick a representative field or fields to count and should possibly be done blinded researcher as to the experiments (IMO). Of course many people use this methods for counting and picking multiple fields from each section helps in confidence but it still does not represent the whole population as can be seen by flow. However, I am by no means suggesting that microscopy is not a valid/ reliable method. All methods will have their upsides/ downsides.

Hope this clarifies.

S

Life Technologies, Live/Dead Viability/Cytotoxicity kit,

:green-fluorescent calcein (live cells) , red: Fluorescent ethidium homodimer-1 (dead)....may be used with flow cytometry , fluorescence microscopy and fluorescence plate readers. Cost: $367

What technique you use depends on what you are interested in. Flow cytometry gives objective answers on large populations of cells but the cutoffs are commonly arbitrary - the majority of 'populations' are enclosed by boxes spanning at least one, sometimes 2 logs. If I found two cells that differed by a hundred fold in their content of a particular marker, I am not sure i would assign them to the same category. The second main question one asks of flow cytometry data is 'to what extent is the number of cells influenced by the preparatory process?' For some studies, this is not an important question, for others, the issue of whether the cells were dead before the preparative procedure or were merely susceptible to the stress of this procedure is important.

It is well known that the refractive index of a dying cell gradually approaches the refractive index of the cell culture media. As mentioned above, this can be observed in a phase contrast microscope.

Quantitative phase contrast microscopy is a novel microscopy technique which can quantify what can be observed in a conventional phase contrast microscope. It allows cell proliferation and cell viability to be non-destructively measured over time, directly in the culture vessel without using stains.

For more information see the attached link.

http://www.phiab.se/technology/quantitative-phase-contrast-microscopy

To have a quick answer, you can use classical Trypan blue and malassez counter and a classic microscopope. But a staining with CFSE or with propidium iodide and cyclin A could be easily done on flow cytometer.

I hope that it help you.