How to run an NC-202 demo for counting cells on microcarriers

Background

Counting cells on microcarriers is a difficult challenge for everyone working in this field. Automated cell counters cannot provide a solution. Usually microscopes or other time-consuming preparation methods are used to count cells on microcarriers. These methods are very complicated, time-consuming, non-robust and results significantly varies between different samples/users. 

We have created an exclusive application to solve this challenge and generate a robust method to count cells on Microcarriers using the NucleoCounter family.    

How To run an NC-202 demo for counting cells on microcarriers

  • First you need to know that this will differ from customers to customers depending on the used methods/Culture and he wants to see.
  • The aim is to detach the cells from the microcarrier surfaces, thus the lysis buffer solution lysis 1.
  • Please ask the customer to have enough samples in order to perform replicates and if it is needed linearity.
  • Some customers would ask about aligning or comparing the results with another counting method. The answer is always, not logical and possible, what is important is the precision and reproducibility produced by a counter which allow us to trust its results.
  • A general, step to step application notes for microcarriers can be found in here:

Using Cassettes:

https://marketing.chemometec.com/acton/attachment/21287/f-0167/1/-/-/-/-/994-0221%20v%201.2%20Tech%20Note%200221%20Count%20of%20Microcarrier%20Cultured%20Cells%20%28NC-200%29.pdf

Using A2 slides:

https://marketing.chemometec.com/acton/attachment/21287/f-016a/1/-/-/-/-/994-3815%20v%201.0%20Tech%20Note%20Count%20of%20Microcarrier%20Cultured%20Cells%20using%20A2-Slide%20%28NC-250%29.pdf

Set up a protocol for demo:

In principal, you need to add solution lysis 1 to the microcarriers sample, suspend and incubate for an indicated time, then let the microcarriers to sediment for an indicated time. Finally, counting using the protocol ‘’C&V Lysis-Count’’

Important facts:

How to decide for how long shall I incubate the microcarriers with the solution lysis 1?

It is a very new lysis solution and rarely tested with the microcarriers. Our last test, a 30s incubation at RT was more than enough, still it may be different between each user. You shall advise the user to make a comparison study while incubating for different time (recommended 4 different time points: 30s, 1min, 1min30s and 2min). At each time point a sample of the microcarriers shall be taken after incubation and observed under optical microscope. This observation will allow us to verify the best time for incubation for all microcarriers to be cell free.

Please notice that a longer incubation time with the solution lysis 1 would affect the general nuclei count. Therefore, the timing should be kept exact for each sample to maintain a good reproducibility.

How to decide for how long shall I let the microcarriers to sediment after lysis?

Once you have achieved to find the best time of incubation with the solution lysis 1, it is mandatory to let the microcarriers sediment in order to have a supernatant of cell nuclei only.

Since there is different type of microcarriers and it could differ in materials and Mass, the speed of sedimentation would vary correspondingly. But what is for sure, is all microcarriers are heavier of a nucleus and would sediment much faster. Also, the sample volume would for sure affect the sedimentation time 

In order to eliminate the sedimentation of the Nuclei also, you have to make sure that you are waiting for the right moment before taking out your supernatant.

Here you have to consider different time points to be tested (We recommend 6 different time points for a working volume of 1 ml: 30s, 1min, 1min30s, 2min, 3min, 4min)

After each sedimentation time point, you have to look at the bottom of your assay tube to see if a visible pellet is form, which is an indication for the sedimentation of the microcarriers. Additionally, you can separate the supernatant formed, resuspend it and look under optical microscope for the presence of microcarriers. In here also, exact timing is important to be considered for each sample counting to avoid strange variations. 

Shall I always aliquot the supernatant (containing the nuclei) in a new assay tube?

We highly advise that to be sure that you are not taking any risks, especially when you have to perform technical replicates from the same sample, where you will be able to easily suspend your supernatant in order to obtain a homogeneous solution all the times.  

Can temperature affect the lysis process?

N/A

Practical notes:

  • Please notice that a technical replicate (5 if possible) is so important to show the customer the robust count and lower CV%. 
  • Additionally, if it is really needed, you can make a series of dilutions to perform linearity test. Attention, it may be tricky in the case of microcarriers.
  • Let different demo participant perform the replicate counting by themselves in order to show them how we eliminated the user bias.
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