Hi
I'm trying a purification kit from stemcell technologies (CD3 release kit). I used 100 million pbmc cells according to protocols. My initial flow cytometry results indicated I had around 38 million cells I only ended up with 20 million cells at the end. Assuming even its 100 % pure isnt that a bit of a waste for purification? you lose so much.
thanks
mostafa
We had the same problem. If you have the data, I would love to document it. StemCell Technologies is aware of it. Their kits are designed for high purity but not high recovery. Recovery of the intended cell type can be as low as 50%.
What was your purity? We used to run both positive and negative fractions through Flow at the end of isolation to check purity/loss respectively. If you see that you have a sizeable CD3 population in your unwanted cell fraction, you could subject them to a second round of isolation. Or you could start with more than recommended of the reagents in your first round of isolation.
StemCell Technology reps are extremely generous in providing technical help and so they could tell you which of these approaches would work better.Greater yield/recovery usually comes with a compromise in purity. You can never recover all of a particular cell subset. This happens with any purification method you use. And recovery goes down with rarer cell subsets.
thanks a lot for the advice Julie. Actually I found there was a problem in our flowcytometer read the purification process was complete. But on a side note, have you guys ever tried down scaling the stemcell technologies kits?
It recommends 100ul for 10^8 cells/ml. Have you guys tried using 50ul or even 25 ul and see its effect on the capacity of purification?
NP. I think the lowest I have used is 80 uL for 10e8 cells. I didn't want to compromise on the purity.
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