How to do a comparison study viable vs. dead cells

Background

Many customers want to test the viability range of our instruments. Therefore, they are trying to kill their cells to get a cell sample with 0% viability. Often, they are trying it via heat shock or non-feeding of the cells, but both approaches are not working. When killing the cells with 70% ethanol and mixing viable and dead cell samples you can generate a comparison study.

How tget 0% viability

Customers usually are trying two approaches:

  1. Heat shock: Wrong – the heat shock will alter the secondary structure of the DNA and DAPI cannot bind anymore to the DNA. In conclusion you´ll see an almost 100% viability
  2. Non-feeding the cells or “leaving them at room temperature overnight”: Wrong – DAPI will only stain the DNA of late-stage apoptotic cells, the cells will be maybe not healthy anymore – but still viable. This will eventually work when you are using trypan blue for staining, as trypan blue in comparison to DAPI will stain early-stage apoptotic cells as well.

For effectively killing the cells we are recommending using 70% ethanol or 80% DMSO. Both approaches work very well. For some cell samples, customers reporting that DMSO treatment would lead to cell clumping. In this solution we will stick to 70% ethanol.

How tdo a comparison study viable vs. dead cells

We do have an excel sheet for performing a comparison study. That sheet can be sent to the customer, all necessary information is already included, and the customer only needs to fill in the columns for total cell count and viability.

  1. Harvest your cells. Determine cell count and viability of your cell sample in triplicates – this is your viable cell sample
  2. Take half of the viable cell sample and make a pellet, discard the supernatant. 
  3. Resuspend the cell pellet in 70% ethanol, mix well by pipetting, incubate for 2 min at room temperature.
  4. Wash the cell pellet with medium and resuspend it in medium. Determine cell count and viability of that cell sample in triplicates – this is your dead cell sample
  5. Mix your viable and dead cell sample in different ratios, e.g. cells 25%:75% und 50%:50% und 75%:25% and determine cell count and viability in triplicates.

6. Enter the values in the excel sheet. The average will be automatically calculated. Compare the “calculated viability” (based on the viability of the viable and dead cell sample) with the “measured viability”.

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