How the meticulous researcher validates their cell counting method

Yes, Meticulous Researchers validate their cell counting method! It’s what qualifies them as being meticulous… Did you ever do it, though?

You can benefit a lot from validating your cell counting method.

First and foremost, if you have confidence that your cell count is correct, you can trust your data and won’t lose sleep over at least THAT parameter of your setup and results.

The Meticulous Researchers know, and we created this cheat sheet to see how they work.

1. They know the difference between accuracy and precision

These are the basics to be on top of in cell counting. Suppose you want to validate the precision and accuracy of your cell counting instrument.

In that case, it is best done by validating the linearity of your cell count through a 1:1 dilution series and ensuring that your results are linear, i.e., that your R2 is close to 1.0.

Cell Counting Method that are both accurate and precise (left), only precise (middle), and neither precise nor accurate (right).

2. They know their working range and linearity of viability

Illustration showing how to determine the working range and linearity of the viability of a cell counter.

Meticulous Researchers have tested the outer boundaries of cell concentrations at which they can count cells in a linear range, i.e., count with confidence.

They have also mixed living and dead cells at different known ratios and tested whether the instrument actually identifies the cells as living or dead.

3. They have tested the instrument-to-instrument variability

When using different cell counting instruments across the production and quality control processes, it is crucial that data validated at one point can be compared to data at a different process point, so you can get an overall picture of the production run and validate the performance.

The Meticulous Researcher did this in their instrument validation through extensive replica testing. Boring, yes. But extremely satisfying when you just KNOW that your instruments are aligned.

Illustration showing how to determine instrument-to-instrument variability. The same sample is counted using different instruments.

4. They know their people’s strengths and weaknesses

Illustration of inter- and intra-operator variance when using the NucleoCounter® NC-202™
Meticulous Researchers know whether one lab member counts differently from the other and whether this is due to sample handling, instrument operation, or something else. They have tested it, they analyzed the data, they know. They just KNOW! Inter- and intra-operator variation can kill a production run.

5. They know their instrument’s strengths and weaknesses

The Meticulous Researcher also knows whether any contaminants of their cell culture production run potentially affect their cell counting method. Yes, of course, they tested this as well – they test everything!
How to test whether contaminants affect a cell counting instrument

Get the webinar on refining your cell counting method

Our Field Application Scientists (FAS) get questions from many customers on how to validate their cell counting method. Luckily, they know how the Meticulous Researcher does this. We hosted a webinar with a full guide, so you can follow the same methodology of the Meticulous Researcher: How precise are your cell counts? It’s not hard at all, it just takes time and dedication, and you will know all about how much you can trust your cell counter.

Further reading

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3 Comments

  1. Nice post, I am using DAPI dye to discriminate living and dead cells in flow cytometry. Currently, I heat-treat cells to have a positive control for dead cells. Unfortunately, I realized that heat treating the my cells creates a lot of debris. Do you have a better way to kill cells?

    Reply to this message
    • Hi Alexander,

      Thanks for taking the time to read through this and leave a comment. We normally recommend killing your cells either via Ethanol fixation or with DMSO. Heat treating not only creates a lot of debris but can cause DNA degradation which will prevent DAPI binding, as DAPI is a DNA intercalating dye. Hence we always recommend Ethanol or DMSO to kill the cells, just be sure to wash out the DMSO afterwards if that is the method you choose.

      Reply to this message
      • What percentage of ethanol or DMSO do you use? Is there a protocol available?

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