Hi, colleagues.
Unfortunately I have just started to work in this new field for me, PBMC purification from blood bags, MACS separation etc.
So what I do is out of a blood bag from hospital I do a ficoll to get PBMCs, then, using PAN T Cell Isolation Kit from Miltenyi (#130-096-535) I am trying to purify T-cells. The problem is that sometimes in terms of cells count number I have something about 50% of PBMCs total cell number, sometimes it's 15%...
Is that normal? I agree, there's a donor variability, but is there maybe some "average" cell count that can be considered as "logic"? If not what should I do? Re-label and do another MACS separation?
Just in case I count on Malassez hemocytometer..
many thanks in advance!
Normal T cell count in PBMCs is around 60-70%. Under normal conditions after ficoll separation and purification by Miltenyi kits, you should get around 50-70% of T cells with purity more than 96%. If you are getting around 50% it is not that bad but you can certainly improve upon your purification process by increasing washing steps etc. You should also aim to process in a way that cell death is minimized.
50-70%% of all peripheral leukocytes, i. e. 1.500 - 4.000/mikroliter blood.
You should distinguish between what you get after Miltenyi-MACS-ing (this may vary considerably) and what is the percentage of lymphocytes in the untouched blood of your donors.
The results you get from the PAN T cell Kit will depend a lot on the quality of your cells. So it would be worth trying to get a good reproducible Ficoll prep which gives you the normal 50*70% of Pan T cells in the PBMC before trying the magentic separation.
I would do a few checks before getting to conclusion.
1-PBMCs number: Even with donor variability you should have a good idea. I usually get around 1e6 PBMCs/ml of normal blood donor.
2- PBMCs viability: If using PBS as diluting media I get above 92-93%, if using HBSS as diluting media that number is slightly higher, usually 96-98%. Avoid dead/dying cells are they are a nightmare when you do a column separation. They will release DNA and clump to viable cells, preventing an optimal separation.
3-Efficiency of your separation: Always do a staining to assess Pan T cells number in your PBMCs pre-separation and post-column. This will give you the efficiency of your separation and, also tell you more about your donor and determine if indeed your variations are due to 'donor variability".
4-Last but not least, the size of your column matters greatly. A good guideline for me is to follow Miltenyi instructions for column size with a twist: never max out more than 1/2 of their recommended binding capacity.
I hope this help.
Good luck
After many primary human samples, both normal and with cancer, the range is very sample specific. but somewhere near and close to the levels mentioned above
Great, thanks a lot for the answers!
But what if I have around 15% of PAN-Ts? Does that mean the separation didn't work? I forgot to mention that we're using QUALIFIED blood bags, meaning donors are healthy.
And once again, when using that 130-096-535 kit, we're doing the NEGATIVE selection - meaning "everything that's not retained on a column (flowthrough) is a target cell population (PAN-T)"..
That seems very low, even for a negative selection kit. How old is your blood? Another step you might want to add is a RBC lysis step to remove unwanted RBC. Using a regular hemacytometer is not enough to define the population, especially if there is poor separation efficiency. As others suggested I would phenotype your samples (CD3+,CD14-,TCRab,TCRgd) at each step to see where you are losing your T cells. And have you analyzed the positive selection fraction to see if there are any T cells?
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