How and when to use Method 2 Assay


The Method 2 protocol should be exclusively used for CHO cells cultured in bioreactors for long periods of time. There can be other cell types effected as well but usually you will have that problem with CHO cells.

When CHO cells are dying, they will degrade and loose their DNA content over time. As we are using DAPI as a marker to detect dead cells, we cannot detect any DAPI signal in those cells anymore using our standard Viability and Cell Count Assay. The viability would be very high even most of the cells are already dead. Those dead cells without DNA content are called “ghost cells”. 

A very common support case is, that customers do not rely on the NucleoCounter viability anymore because they get a viability of 99% after weeks in culture when using the standard Viability and Cell Count Assay – in comparison to that, their trypan blue method will give them 60% viability. This is a clear sign that the reason is the degraded DNA and they should switch to the Method 2 Assay.

The difference between the standard Viability and Cell Count Assay and the Method 2 Assay is only the algorithm analyzing the results. The algorithm will lower the threshold for the DAPI signal, meaning even cells with low DNA content can be recognized as dead cells and the viability will be displayed correctly when using the Method 2 Assay.

How to run and analyze the Method 2 Assay

Running the Method 2 Assay requires gate adaptions. And those gate adaptations are dependent on the culturing time as well. That is why it is important to ask the customers to run different cell samples for different time points of the culture with the Method 2 Assay, meaning from seeding until harvesting the cells. Only after looking at all these cell samples you can adapt the gates correctly.

How the gates should be adapted:

  1. By default setting, cell debris are included. A new protocol should be generated to exclude the cell debris by closing P1 gate.

2. Optional: Sometimes, the cell population on the left side is not completely included into the P1 gate. Those are the live cells with a very low DAPI signal. Make sure, the gate is opened completely.

  • Open the first plot and click on the x-axis. Largen the zoom area to the left by increasing the number.
    • Click enter. Now you can see the whole cell population. Open the P1 gate and include the whole cell population. And of course, take care of the cell debris as well.
    • Optional: Save it as a new protocol.

3. Optional: The discrimination between live and dead cells is not clear, check different time points of the sample and adjust the P2 gate.                       

How to reanalyze results with the Method 2 Assay 

If a customer used by mistake the standard Viability and Cell Count Assay for CHO cells, there is often a need to reanalyze the results using the Method 2 Assay. Reanalyzing those results is time-consuming and quite difficult as there are many pitfalls when handling the software. Try to figure out if the customer can do it by themselves or if you need to do it to ensure good quality and service.

  1. Reanalysis:
    1. When trying to reanalyze results with the Method 2 Assay you will notice that the “reanalyzed result” only contains a small table with the new results. Why are there no plots? Because in the default protocol the option “show count gates in plot manager – No” is selected. But as the Method 2 Assay needs protocol adaptations as mentioned above, it is necessary that you get the plots as well. 
  1. To avoid this, you need to create a new protocol and choose in the protocol adaptation wizard in step 3 “show count gates in plot manager – Yes”. But creating a new protocol is only possible if you´ve already gained results with the Method 2 Assay. Please find attached 3 protocols for reanalysis (for the NC200/ NC250 and NC3000) as well as some raw data gained with the Method 2 Assay.
  2. To reanalyze a result, choose the reanalysis protocol in the protocol folder and click directly right on the result (wrong: clicking first left and then right). Choose “reanalyze image file with selected protocol”.
  3. Now the small table should pop up (Figure 6) and the plot manager will open and show the dot plots for that measurement.
  4. This should now be repeated manually for each single result you want to reanalyze.
  1. Gate and protocol adaptations:
    1. When all results are opened in the plot manager, adapt the gates (see above) with special attention to excluding debris, including all cells in P1 and distinguishing between live and dead cells.
  2. Save all results.
  3. Create a new protocol for the customer with all those adaptations.

3. Optional: Presentation of the results in an excel sheet to show the customer how the viability and cell count is changing over time when using the correct assay

APPENDIX: Raw data and protocol can be found here:

R:\KB\Appendix to How to entries\How to and when to use Method 2  

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