Hoechst dye stains the nuclei even of living cells, there is no need for fixation, but it can of course be done, I am staining living cells as well as fixed cells with it by using Hoechst 33258. But after staining cells for something like 30-60 min you have to wash cells. Afterwards you can easily go for microscopy, the advantage of staining the nuclei is that you can see if nuclei are damaged and cells will finally dye.

I don't think that you will really find any staining marker that you can use without washing steps as you give it in abundance and you have to remove it as you otherwise have too much background.

Daniela, thanks for the answer: Sorry my question was not clear, but I am looking for a differential stain. I would like it to stain ONLY the living cells while leaving the dead unstained.

Best... m

So maybe you can check for Propidiumiodide or 7-AAD, but this would be the other way round - only dead cells make an uptake of Propidiumiodide and 7-AAD whereas living cells with intact membrane will not be stained.

Both are commonly used in combination with Annexin V in flow cytometry to distinguish in between living and dead cells...

Thank you, Daniela. There are indeed some exclusion dyes for which this process works. However, as you fix the cells they die and often these newly dead cells also take up residual dye leading to a population of faint and strongly stained cells. Couple that with nuclei already fluctuating in brightness due to variable chromatin condensation and heterogeneity in the cell cycle stage and the distinction between live and dead is not that clear anymore.

Calcein AM could work for you. Ideally, you should wash the wells but it is possible to find the write conditions that wash is not necessary.

Barak, if I understand right Calcein AM does not allow for fixation. In other words, if the membrane gets perforated (is it inevitably does with formaldehyde fixation) the dye leaks out of the cells.

Dear Mario,

your questions sounds interesting but it is for answerers BAD formulated! WHY?

At first please give nore infos about your cells in the well.P.e monolayers (HeLa, fibroblastes) or 3D growing cells like tumor cells. In this case, you stain only the surface of the tumor cells.

Concerning your requirements:

1) Staining should be possible by simply adding a solution to my wells and waiting.

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I am a retired old microcopist and worked with dyes and fluorochromes for vital staining between 1968 to 2000.

Cationic dyes are toxic for cells if the concentration is too high.

Please note that you need to know the concentration of the dye or fluorochrome used for the vital staining. Between 10-4 and 10-7 you can see for some dyes the letal effect.

You must know if the choosed dye accumulates in the lysosomes or stains the mitochondria p.e. prior to further staining.

Waiting is a problem if your cells are very sensible to the action of the dye.

Please test the staining after 15 mn, 1 h ....

2) It should not be necessary to remove the remaining dye/staining solution or wash the cells.

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You cannot observe anything if you have the solution in the well has a own fluorescence.

3) The dye should be retained by the stained cells even if I fix them with formaldehyde.

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Please more infos about the modus of fixation with formaldehyde (Protocoll)

4) your answer :

Thank you, Daniela. There are indeed some exclusion dyes for which this process works. However, as you fix the cells they die and often these newly dead cells also take up residual dye leading to a population of faint and strongly stained cells. Couple that with nuclei already fluctuating in brightness due to variable chromatin condensation and heterogeneity in the cell cycle stage and the distinction between live and dead is not that clear anymore.

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You can stain cells and reproduce the staining ONLY if you know :

- How old are the cells (How many passages). Please infos.

The fluctuations can occur if you have a different population of the cells and at different stages in the cell cycle. Some cells allow ONLY a limited number of divisions (related to your remark/answer about fluctuation).

- The number of dead cells doesn 't give a complete picture of the changes occuring in a aging cell population. Too many parameters are unknow in your project!

Please answer and good luck.

JRG

Tamara, as with many other dyes, even though it is called a 'Viability Dye', the eFluor 450 stains dead cells.

Jean, thank you for your answer. I will try to answer your questions better, just in case you can help me out further.

I am looking for a protocol that will work on a wide variety of cancer cell lines growing in a mono-layer on plastic dishes.

1) The amount of time to 'wait' is dependent on the dye I use. I would prefer to fix the cells after an hour maximum since otherwise I worry that toxic effects of the dyes alter my results. If the dye stains the live cells within minutes, I will fix within minutes.

2) That is true. However, a lot of dyes change their fluorescence intensity due to either enzymatic activity , pH/redox of the stained compartment, or binding to e.g. DNA. For example Hoechst dyes give perfectly acceptable staining results even if the dye is not removed from the media.

3) I am perfectly open to any form of fixation protocol, as long as it does not require removal of the media in the plate. Currently I am fixing by the addition of formalin to a final concentration of 3%. But I have also used PFA to the same effect. I went with formalin since it has a higher concentration and is much cheaper. Since I am not doing any antibody staining the results with formalin have been quite consistent.

4) I use my cell lines for maximally 25 passages and then thaw a new batch of cells. The overall results have been highly repeatable within these parameters. My problem is not that I have difficulty reproducing my data. I am merely trying to accurately distinguish between cells that are alive/healthy versus cells that are dying/dead after drug treatments. In many cases there appear to be cells that changed morphologically to dead cells (they rounded up and partially detached from the dish), yet, they still contain a clearly discernible nucleus, so if one determines cell number by simple counting one would overestimate the live population.

The reason I would like to fix my cells (rather than using the typical live/dead stains that do not require fixation) is that measuring all my plates requires up to 24 hours. To ensure reproducibility it has been proven advantageous to fix the cells.

Best... mario

This is my suggestion.

The use of metabolic probes would make hard to perform the fixation step. My advice is to use two DNA stains. One is a permeable DNA stain (I would recommend LDS-751 if you want to use FACS and a blue laser). This dye will stain all the cells alive and dead and the stain will remain even after fixation. The other dye should be a DNA dye impermeable to plasma membrane and will stain only the dead cells. If you want to combine with LDS-751 a good option would be SYTOX green which is green emitting with minimum spectrum overlap..

Thus, alive cells will stain only in red while dead cells will be both green and red. Of course when cells are fixed you should remove the DNA dyes. Staining process is pretty fast with these dyes and you could fix after few minutes from staining, but after washing to remove the free dyes.

I don`t know which is the parameter you are looking for. If it is something related to morphology I guess you have to work on a plate or a chamber slide. If it is an antibody the FACS will work pretty well. This procedure is useless if your cellular model is adherent, since you will find the dead cells mostly as floating.

Cristiano, thanks for the notes. I am using 384 well plates with adherent cells. The problem is that cells that are dead, or dying (due to my treatments) are so loosely attached to the plate that I lose a substantial number of cells during any step that requires removal of the media.

However, on the flip-side, I also need to fix the plates since I have to image so many of them that imaging without fixation would give drastically different results between the first and last plates.

I may give some of the MitoTracker dyes a try. They supposedly can be fixed and only stain live cells. While they suggest removal of the dye following staining, I may give it a shot to use a low dose and not remove it during fixation.

I worked with mito-tracker in the past (PMID 9450528).

I would not recommend such approach since you will stain both alive and dead cells and after fixation you will have a precipitate in the cytoplasm which will not allow the use of simple image analysis algorithms to detect nuclear staining. In dead cells you will have a staining, although with a lower fluorescence intensity and when you will remove the dye you will have diffusion of the staining from the mitochondria.. I feel the protocol I suggested should work pretty well in 384-plate through imaging and cell content analysis or a "cellomics" like based assay if you can get access to this platform. You can gate out all the cells with green fluorescence (dead) and keep the only red as the population of interest to analyze what you want in a single plate.

Dear Mario,

thank you for your fast answer. I need still some infos before I send further answers.

- Are you using fetal bovine serum (FBS) for your cell culture or serum free media?

- Do you wash the cells prior staining with the solution without dye?

- You don 't answer the question about the concentration limit of your dye at first. With very low concentration 1x10-7 or 1x10-8 , you can obtain different ways of the staining.

A good HINT : Test for dye uptake from 5 mn to 1 h.

It is worthwhile to make a test a staining with a dye e.g. fluorochrome via fluorescence microscopy. With very slow concentration , you can follow p.e within 10 mn the diffusion of the dye/fluo through the cytoplasma membrane ( then the lysosomes staining) then the transport through the nucleus membrane and finally the nucleus staining .

The synchronization of your cells may be interesting if you want to have cells in the same stage and you must stain the cells in the same time because cells live its own cellular rhythm.

Pictures as infos for transport during staining: ( Lab Research 1996)

I send you 2 pictures of a stainng of HeLa cells (C= 0,1 microMol) with an imidazoacridinon dye C-1311 (Fluorescence micrscopy).

The strong uptake during the cell division can be seen. The uptake occured within 10 mn through cytoplasma and nucleus membranes with this dye.

Details upon request.

Good luck!

JRG

Jean, I am indeed using FBS in my culture conditions and I can not wash the cells prior to the application of any stain. The loss of loosely adherent cells is too large to obtained reliable and reproducible cell counts.

I am certain the the concentration limit of dyes depends mostly on the dye being used. (both for the lower and upper limit). I have not really tried many, so I can't answer this question. For Hoechst, clearly this is not an issue since I add the dye concurrent with the fixation solution and it has such a high differential between it's unbound and bound state that even very high concentrations can be readily imaged without removing the stain from the media.

I'd be happy to try the staining conditions you suggested. Do you know if this a) this stain is fixable and b) it differentially stains live cells over dead cells?

Thanks... m

Hi Mario,

I believe your best bet would be to use both and live and dead cell stains together which are fixable. Molecular probes has these which could be directly added to your culture media and these are well retained after fixation too.

http://www.lifetechnologies.com/order/catalog/product/H10290

Dear Mario,

 I think you can use cell trackers available from molecular probes and are easily taken up by live cells and are fixable. they have brilliant fluorescence even after fixation and are retained in the cells for 7 generation. But they need to be used in serum free media and i think 5uM should be sufficient to stain the cells. you have to incubate in dark at 37 deg C for 30-40 min with cell tracker diluted in serum free media and then remove the media and wash gently once and add back complete media or fix them as the case may be.

I hope this helps.

Hither

i use cell tracer Vybrant green at the high conc allowed according to manufacturer I leave cell in pbs containing the stain for 15 min the leave in fresh medium few min the I add them to 1% fibs medium but 24 hr are the dye leake and could only capture bf imageI neeed advice

Dear jean,

can you please suggest dye to stain bacterial cells and its progeny without affecting its growth in media.

thanks