Hi,
I am working with HMC-1 cells and I want to measure live/dead cell numbers after treatment but for very short time (i.e. 5 minutes up to one hour). This may be tricky since these cells are floating cells and I have an idea on how to perform this study but I have a few questions:
1. seed cells in 48-well plate at 5x10^5 cells/ml
2. let rest few hours
3. add treatment at desired dilutions
4. after each time points, can I take an aliquot of cells (i.e. 10uL) directly out of the well and and mix with trypan blue at 1:1 dilution to count cells? Should I count each replicate separately to get an average of live/dead? I plan on counting using the tC20 automated cell counter, is that reliable for this method?
Does anyone know of a way to stain these cells with trypan blue for example to visualize cell death over short time?
Thank you.
Good Day!
1.If these cells are floating, you can take 10 μl of the suspension and add trypan blue there.
2. To obtain the average value of cell death, it is necessary to count each repeat separately.
3. I had problems with TC20, because in the protocol the boundaries of cell sizes were constantly getting confused and if there were large or small cells or their clusters, then TC20 did not take them into account. For a rough estimate, you can of course use TC20.
Sampling at short time points: Yes, you can take small aliquots (e.g., 10 µL) directly from the wells at each time point and mix them 1:1 with trypan blue for viability assessment. Just ensure gentle mixing to avoid introducing air bubbles or mechanically stressing the cells. It's definitely best to count each replicate separately. This allows you to calculate an average and standard deviation, which improves the reliability of your results. Make sure to consistently sample from the center of the well, as floating cells may distribute unevenly. The tC20 cell counter should work fine with trypan blue-stained cells, although its sensitivity can vary depending on cell size and clumping. A good alternative that I personally use is the LUNA™ III ( https://logosbio.com/luna-lll/ ), which offers high-resolution imaging and more accurate discrimination between live and dead cells—even for suspension lines like HMC-1. It also supports both trypan blue and fluorescence-based viability assays (e.g., AO/PI), which might give you better precision for short time-point studies. Trypan blue is fast but does have limitations in sensitivity during early stages of cell death. For higher accuracy—especially at 5–15 min time points—you might consider switching to fluorescent viability dyes (e.g., AO/PI or calcein AM/PI). These can detect early membrane integrity changes that trypan blue might miss.