What’s the problem with staining cells for analysis?

To stain or not to stain…you no longer have to choose as label-free cell analysis is here

If you haven't worked with cell stains or dyes before, you might be wondering: Why do we need to use them and what's the problem with using dyes anyway?

Why stain cells for analysis?

Very simply, cell staining is required to perform cell counting and viability assays which are key metrics in cell-based assays. They enable us to examine the cell's overall health and ensure quality control in cell manufacturing processes (1).

Researchers need to count cells to monitor the doubling time and for quantification. Quantifying cells is essential in standardizing experiments that require accurate and consistent numbers of input cells like cell transfection and quantitative PCR. In cell culture, it helps determine the confluence level before diluting the cells into smaller aliquots for optimal cell growth (1).

What’s the problem with staining cells?

Well, getting straight to the point, cell staining has three major disadvantages:

• Its time-consuming: depending on the kit or dyes used, it can take up to one hour to complete sample preparation.

• It can affect cell functionality: The reasons behind this are becoming more clear as we begin to understand what these stains/dyes do to the cells. If your cells are plentiful and you don’t need to recover your cells for further experimentation, then this doesn’t really matter. But if you do need to recover them; then this can be a huge problem.

• Time-point at which cell stains are used for analysis is just too late: a perfect example of this is confirming transfection efficiencies.

Depending on the assay you are carrying out (e.g. cell counting, cell viability or transfection efficiency), there are different stains:

1. Cell Counting commonly uses DAPI (4', 6-diamido-2-phenylindole) and Hoechst which stain the nucleus of both live and dead cells, which then appear blue.

2. Cell Viability Analysis – to differentiate between live and dead cells – commonly uses a number of different stains:

• Trypan blue (TB) – this dye penetrates dead cells membrane coloring them blue. TB use raises environmental and health concerns due to its potential teratogenic effects (4).

• Erythrosine B for dead cells is a dye that stains pink non-viable cells with disintegrated membranes. It is less toxic than TB (4).

• Calcein AM, a green stain for live cells.

• Propidium Iodide, a red stain for dead cells.

3. Transfection Efficiency Analysis – typically uses stains that target the cell receptor or protein expressed by the transfected cell. These assays can only be conducted 24-48hrs post-transfection after plating the cells for culture. This is a huge bottleneck in optimizing transfection efficiencies since researchers do not know whether their cells have transfected or not until they confirm the expression after 1-2 days. Why? Well, because the very method by which cells are transfected involves disrupting or opening the cell’s membrane to enable the vector or gene insert to go inside the cell. This is where the crux of the matter lies and why cell staining doesn’t work! Ideally, researchers only want to plate the cells that have successfully had their membranes opened as these are the only ones that can possibly have the gene insert delivered inside the cell. But the cell stains mentioned above, would actually go inside the cells indicating that these cells are dead – but they’re not. In fact, these are the very cells that you want!

No wonder researchers are frustrated. All traditional cell counting and viability analyses use cell staining methods and the most common methods are:

• The hemocytometer – a thick glass microscope slide with a grid in the middle. With the help of a clicker, you can count the cells within a square and determine the number of cells in a volume of solution (3). Although widely used, the method is laborious and time-consuming.

• Flow cytometry – it involves running cells labeled with fluorescent stains through a flow cytometer. This method can be costly because it requires complex equipment (3).

• Image-based analysis – light microscopy is a less expensive option than flow cytometry. Still, it requires handling large amounts of image data, which can be limiting. Since each instrument has a specific chamber, slide, or cartridge, end-users are tied to every assay's high cost of consumables.

Label-free Cell Counting and Viability Analysis

But what if there was a way to count and analyse cells automatically without using dyes? The good news is this technique already exists.

Cellix presents The Inish Analyser : an automated method for cell counting, cell viability analysis and transfection efficiency prediction based on impedance spectroscopy.

The workflow is pretty straightforward. You put your sample in a tube with the buffer, put the tube in the device, and follow the instructions on the touchscreen. The Inish Analyser gives you an automatic cell count or cell analysis in minutes and that includes the sample preparation!

Unlike laborious manual cell counting, image-based analysis, or flow cytometry, the Inish Analyser requires no cell staining, reducing the steps in your workflow. This method helps to avoid user-to-user variability for higher reproducibility and accuracy of the results.

Predicting Transfection Efficiency

One of the best features of the Inish Analyser is that it can give researchers a transfection efficiency prediction in minutes, immediately post-electroporation. But what exactly does that mean?

As we’ve already described, transfection is a method for inserting a vector, gene insert or foreign DNA into a cell. The most common method of performing cell transfection is electroporation, which involves applying an electric field to increase the cell’s permeability (5).

After electroporation, your cell sample will contain different cell subpopulations, (5):

• Alive but closed: although they’re alive and probably healthy, these are not useful since they were not opened and therefore could not be successfully transfected.

• Dead,

• Damaged, and

• Alive and open: These are the ones that the researcher wants – as they are the only cells which have opened and can possibly receive the foreign nucleic acid successfully (5).

But as we’ve already mentioned, traditional cell analysis assays can’t differentiate these cells from dead cells since their membranes have all been opened (5). So, viability stains would indicate that these cells are dead when in fact they´re not!

However, with the Inish Analyser, you can distinguish these cells from the other subpopulations. Immediately after performing transfection, cells are taken from the electroporation cuvette and transferred to a sample tube with the Inish Analyser buffer to perform the transfection prediction assay. This allows you to:

• Quickly determine which electroporation settings are working and which aren’t

• Optimize your workflows ensuring that you are plating [alive and open] cells ensuring higher transfection efficiencies.

This also has the added benefits of saving you inserts, media, and more importantly, time.

If poor transfection efficiency is wasting your time, we can help. Contact Cellix to book an online demo or request a quote.

Conclusion

In summary, the cell analysis assays we’ve described above are fundamental to cell biology research in both industry and academia. Working with cells can be challenging and every step is crucial for the experiment's success. But fortunately, the beauty about science and technology is that it is constantly evolving. Cell counting, cell viability analysis and transfection efficiency analysis are also evolving to more practical and less expensive ways. The Inish Analyser is the latest offering to the scientific community which is a highly cost-effective option for your lab as it improves workflows and reduces working hours and the consumption of consumables and reagents. If you’d like to learn more, contact us now to book an online demo or request a quote.

References

1. Bio-rad. Cell counting methods [Internet]. [cited 2021 Aug 7]. Available from: https://www.bio-rad.com/pt-br/applications-technologies/cell-counting-methods?ID=LUSOLB470. Access: 08/07/2021.

2. Technology CS. Synopsis of Cell Proliferation, Metabolic Status, and Cell Death.

3. Fiala J, Lloyd DR, Rychtera M, Kent CA, Al-Rubeai M. Evaluation of cell numbers and viability of Saccharomyces cerevisiae by different counting methods. Biotechnol Tech. 1999;13(11):787–95.

4. Kim SI, Kim HJ, Lee H-J, Lee K, Hong D, Lim H, et al. Application of a non-hazardous vital dye for cell counting with automated cell counters. Anal Biochem [Internet]. 2016;492:8–12. Available from: https://www.sciencedirect.com/science/article/pii/S0003269715004406

5. Chong ZX, Yeap SK, Ho WY. Transfection types, methods and strategies: a technical review. PeerJ [Internet]. 2021 Apr 21;9:e11165–e11165. Available from: https://pubmed.ncbi.nlm.nih.gov/33976969