I isolated NK cells from PBMCs with MACS buffer and NK cell isolation kit (Miltenyi Biotec).
Firstly, I did ficoll separation, and then I did NK cell isolation following the manual of the kit.
Although I experimented following the same protocol, I wasn't able to do isolation well.
I attached a PDF file of this result (Each sample is from another person).
From left to right, NK cells isolated with new LS column, NK cells isolated with reuse column（2nd-time）, NK cells isolated with reuse column（2nd-time）
Would you like to tell me about my mistake and what should I do next time?
It's not clear from your description what the difference is between the middle plots and the plots on the right side.
I don't isolate NK cells by MACS, but I do isolate CD34+ cells from human cord blood on MACS. I never reuse columns.
I have tried sequential isolation to separate subsets of CD34+ cells, and in my experience, the 2nd isolation is never as clean as the first, but that could be a problem with the releasing step in between isolation #1 and #2.
Gary Lee Gilmore
Thank you for answering!!
The middle plots are from a person and the plots on the right side are from another person. Each plot is the result of the experiments conducted on another day.
Sorry, I couldn't understand "sequential isolation".
Would you tell me about that?
I didn't skip any steps.
Hello, Arisa!
Please You look at following protocol:
Article Expansion, Purification, and Functional Assessment of Human ...
Isolation and Characterization of Human UNIT 7.34 Natural Killer Cell Subsets
Article Isolation and Characterization of Human Natural Killer Cell Subsets
(attached)ISOLATION OF HUMAN NK CELLS FROM PBMC
Alexandr Chernov
Thank you for answering!!
I didn't know Rossetesep human NK cells!
I want to refer to this protocol.
Ah, so that would explain why the middle and right columns had differing input % of the cells of interest. Patients - and normal controls for that matter - can vary greatly in blood lineages depending on a number of factors, but especially disease state. So I would not typify your result as a failure, but as a variation due to the different person.
The protocol posted by Alexandr Chernov looks to be helpful.
Sequential isolations can be performed by MACS with specific reagent kits - ones that are designed for microbead release to permit secondary isolation. The prototypical system is for isolation of human CD5+ B lymphocytes. Step 1 is a CD19 MACS isolation to enrich for B lymphocytes, followed by a release treatment and wash, and a second MACS step using CD5+ microbeads.
I was attempting to isolate a CD19+ subset of human CD34+ cells. In 3 attempts, I got ~ 40% pure with ~ 50% yield, never much better than that, so I dropped the project.
Gary Lee Gilmore
Thank you for telling me very politely!!
I see. I got that the result seems like a variation due to the different person.
I understood that MACS is used in that way.
I have another question.
How pure is NK cell preferred generally?
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