What is the best method of neutrophil isolation and anti-coagulant if I want 20 millions of healthy and unactivated cells in 95% purity?

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Javier Garcia Palomo

Try the Ficoll method plus cells sorting in ACD anticoagulant. In the first case, use the pelleted cells

In order to sort the cells we are using Automacs with antiCD15 beads. With this metod i have reached >99% of cell purity

Ada Tse

Thank you very much for your information but we do not have a budget for this luxurious method. Perhaps in the future!

Olivia Ng

From my optimisations I'm getting about 14 million viable cells from 20ml of whole blood using the ficoll then dextran method. Purity is usually >92% if you don't do a CD15 bead sort.

@Olivia Ng That sounds good. I asked people who used Ficoll but the purity is not so good. How did you check the purity? And is it possible to send me your protocol? Thank you!

I check purity using CD15 and CD66b staining by flow cytometry. Other people will advise you to do Giemsa/Leishmann staining or look for MPO activity. Let me know your email address.

Vartika Sharma

Olivia Ng Hi, I also do something like this for neutrophil isolation but neutrophils are getting attached to the surface. Can you send me the protocol for my reference? Thanks a lot.

Vartika Sharma can you clarify what you mean by getting attached to the surface? Are you using Ficoll?

Olivia Ng , Hi I meant after isolation when I am keeping them in the tissue culture plate. They are immediately attaching to the surface of the plate. Yes I am using Ficoll.

Vartika Sharma they are sticky cells, more so if the viability is poor. Also please check your cell density when you're plating. If you send me your email address, I'll give you the protocol.

[email protected]

Cells were viable as I did Flow Cytometry on those cells. It is just that I had to scrap them off before proceeding for the staining, which I know is not a good idea as it activates the cells. And I require unactivated cells. I am repeating the experiment with few more modifications. It would be great if I can have a look at your protocol.

Thank you