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Thoughts on best ways to determine cell concentrations?

Posted By: Winegard, on Dec 9, 2016 at 1:05 PM

Hello 10X'ers.  We've been very excited by the early data we are getting on the 10X.  Thus far we have run some experiments with basic K562 cells, primary multiple myeloma cells and mouse islet cells.  For the most part, everything looks good (all with the v1 chemistry so far - v2 just arrived today).  However, one interesting thing we have seen is that we are often off on our cell estimates.  We currently count by using a Countess automated cell counter and then the handy 10X chart to determine the volume to add.  However, we see quite a variation in the number of cells identified post sequecing.  In some cases the numbers are smaller (4500 cells vs 6000) and sometimes they are quite a bit more (2500 vs 1200).  I suspect actually that it is not an encapsulation issue but rather more that we are not estimating our cell numbers accurately. We are going to start to do manual haemocytometer measuresments alongside the Countess for the next few samples and see if that improves things.  We've heard from peole doing Drop-Seq that they find the manual method quite a bit more accurate.  Just wondering what others using the 10X have found?

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Re: Thoughts on best ways to determine cell concentrations?

Posted By: cheny79, on Jan 27, 2017 at 4:40 PM

We are using ViCell machine to detemine cell density. It is accurate for most samples (various tissue types), however toward lower rather than higher numbers. For example, when we targeted 6,000 cells, we got from 5,500 to 4400 cells depending on cell types and sample prep. I suppose this is due to sample loss along the process (their so-called 50% recovery rate, which in our case is more like 40-45% recovery rate).

Re: Thoughts on best ways to determine cell concentrations?

Posted By: jens-10x, on Feb 21, 2017 at 8:50 AM

 

Having two independent counts for the same sample definitiely will improve cell count accuracy and is good practice.  A hemocytometer is generally more accurate compared to automated methods the smaller the cell size gets.  Also, the cell concentration is important to arrive at an accurate cell count.  Generally, very dilute samples (<100 cells/ul) will have an increased standard deviation and may lead to inaccurate counts, especially if only counted once.

 

Having said that, there are a few more factors other than inaccurate counting that can contribute to discrepancies between targeted and reported cell count.

 

i) Cell viability: high fraction of non-viable cells decrease cell recovery

ii) Sample prep time: some cells tend to clump when left on ice for too long which will decrease the # of single cells in suspension

iii) Pipetting volume/ handling: Loading 2 ul of cell suspension to the master mix and being off by only 0.5 ul will have a significant effect (+/- 25%) on cells loaded.  You can mitigate that risk by lowering cell concentration and loading more volume (i.e. ~10-15 ul).  Also mixing the full cell suspension volume with an appropriate pipette just prior to transfering the cells to the master mix and chip loading is crucial.

 

Best,

Jens