I did my PhD in the Department of Chemical Engineering at Imperial College London. My research focused on mathematical modeling of the cell cycle in leukemia and involved experiments with cell lines. During that time, I had to count cells with a hemocytometer so often to track growth that I got tired and decided to build an app, HemocyTap, and share my knowledge on the topic here to help as many people as possible.

Looking for a live/dead assay, and not sure which one is best? Here we have the answer for you.

Why do a live/dead assay?

You should regularly track the viability of your cell culture, as it is representative of how healthy it is. The way you do this is by counting live and dead cells using a hemocytometer, and adding a dye that will selectively penetrate inside the dead (or apoptotic) cells, whose cellular membrane is damaged, while live cells will stay unstained. It’s really a simple method, and if you are counting cells with a hemocytometer anyway it’s worth to throw in a viability dye and count both populations (after all, most of us have two hands right? So don’t be stupid, count live with one counter and dead with the other one!).

What are the most commonly used dyes?

All viability dyes to be used together with a hemocytometer have the property of penetrating damaged cell membranes. The two most common are trypan blue and erythrosine B. Trypan blue is a blue (…) dye chemically derived from toluene. Erythrosin B is a cherry pink dye composed of a fluorescein salt (i.e. sensitive to light at certain wavelengths).

The duel: trypan blue vs. erythrosine B

Unsure about which one fits your needs best? Have a look below.
Trypan blue Erythrosine B
Stains what? Dead cells + serum proteins Dead cells only
How much do I add?* >400μg/mL >5μg/mL
Time needed since cell death* >50min 1min
In case you’re into fashion, what color? blue pink
In case you drop some, how bad is it? Toxic (known carcinogenic) It’s a food additive, what do you think? Bad for your clothes, maybe.
For cell lovers: how bad is it for the cells? Toxic Non toxic for periods of over 2h
*http://jhc.sagepub.com/content/32/10/1084.long

Overall, erythrosin B is a more reliable, less toxic method both for you and for the cells.

Comments

    1. Hi Isha,

      Quick answer: yes. Explanation: The debris are just cell fragments of dead cells. When they die, cells membranes become permeable to the liquid around them (be it water, PBS or a colored dye). If they are further lysed (degraded), the dead cell breaks and releases its cellular contents. This is what you call cellular debris, and since they already had lost their membrane capacity to isolate them form their environment when they were dead, they do uptake trypan blue, or any other dye. See for example here, the sample contains cells that have been exposed to chemotherapy so they are all dead (stained blue), and you can see a lot of blue particles in the background as well, which are the cell debris.

      Hope that helped!

      Maria

  1. Hello,

    I have a question. When we used the Erythosine-B what happend with the cells that may be are in cell cycle arrest? are alive, so I showld see them unstained?

    1. Hi Laura,
      If they are in cell cycle arrest, they should not be stained. If they become apoptotic (which can happen if you leave them in cell cycle arrest for a long time), they can become stained.
      Hope that helps!
      Maria

  2. Dear Maria,
    Which Erythrosine B dye are you using (supplier, concentration ect.) when counting cells in a hemocytometer. We would like to change from Tryphane blue to Erythrosine B.
    KR
    Sabrina

    1. Dear Sabrina,

      In my lab, we always purchased the already dissolved Erythrosin B from ATCC (ATCC® 30­2404). As the protocol indicates, a 1:1 dilution with the sample works best for staining in cell counting. 10uL of Erythrosin B per 10uL of sample is enough (otherwise you waste a lot!).

      Hope that was useful, let me know if you need further details.

      Best,
      Maria

      1. Dear Maria

        Thanks a lot for you quick reply. Erythrosin B from ATCC (ATCC® 30­2404) is however not avaible anymore it seems. Do you have any other suggestions, that are easy- ready to use?
        KR
        Sabrina

        1. Dear Sabrina,

          Oh right, ok. It seems that all other products I can find are in powder form. It’s not too bad, you can always prepare the solution and store it in aliquots, they should be stable for months. Here’s the link for the Sigma Aldrich product for example. Once the solution is prepared, it should be as easy to use as the liquid form product.

          Best,
          Maria

          1. Dear Maria

            Thanks a lot. I’ll have a look on that one. Would you still go with 0,1% or do you reccomend another percentage in a comparative study to tryphane?

            KR
            Sabrina

          2. Dear Sabrina,

            To prepare the solution, I would go with the liquid volume recommended by the manufacturer to add for the powder in the flask. I never diluted that to then count cells, Erythrosin B is not harmful to them so no further dilution is needed (aside from adding it 1:1 to the volume of sample to count).

            Hope that helps!

            Best,
            Maria

  3. Dear Maria

    I have just counted my algal zooxanthellae cells and now have total values of both live and dead cells using four big squares and diluted each culture sample of 1 mL once. In each count, I have four samples from each of the four replicated cultures, leading to total number of live cells to about 1000 and of dead cells to about 40 depending on the temperature at which they were exposed (each replicate culture has four counts). I need to get cell densities or concentrations and viability under each temperature
    My question is should I total and average number of cells for both live and dead cells per replicate culture to get cell densities and viability or have each of the replicates let it have its own for easy calculations?
    I am failing to attach just a small piece of this data from excel as it gets messed up when pasting it here or maybe I should email it for easy demonstration. Please kindly assist and if more information is needed, I will happily provide it. Thanks

    1. Hi Siviwe,

      You should always take the total from each of the samples you have counted. So I assume you have, say 240, 260, 233, 267 for the live cell counts in each of the four samples, and 9, 11, 12, 8 for the dead cells. You would do 240/(240+9)=96.4%, 260/(260+11)=95.9%, 233/(233+12)=95.1% and 267/(267+8)=97.1% and then use each of the four percentages in the calculations (average the percents for a summary stat per replicate culture).

      Does that make sense?

      Cheers,
      Maria

      1. Great. This is wonderful and I think I now have better understanding after looking at your approach to this case and you made it simpler for me, I was at first getting confused. If I average the four percentages you have calculated I come up with 96.1% which I think should represent viable cells from the four replicates selected. I find the hemocytometer calculator fascinating and wish I can have the software and HemocyTap installed in phone or PC for saving and plotting of results (Cell density, Live cell number and Viability) after calculations. If there is an option for doing this function other than the video clips, please kindly share with me the link or direction. Many thanks Maria

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