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invariant NK cells

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jamesaries

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Posts: 14

Joined: Thu Sep 22, 2016 2:49 pm

Post Thu Oct 13, 2016 10:34 am

invariant NK cells

Dear all,

I'm new to the Cytof so please bear with me. I'm analysing PBMCs post-transplant but currently I'm trying to optimise my panel on healthy samples from frozen.

I have an invariant NK cell marker (TCRVa24Ja18) that I am using from Fluidigm. I have used a protocol that combines the cell surface followed by the nuclear staining protocol (I have other markers too!) and used 1uL of the antibody.

I realise the population is very small (?0.2% of lymphocytes) but I have approx 700,000 cells, so I would have expected to find some positive cells, but it doesn't look like it has worked.

I wondered if anyone else has any experience of using this marker? Have you had to titrate up a lot? Any good positive control cells I could use?

Many thanks,

James
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Oct 13, 2016 3:35 pm

Re: invariant NK cells

Hi James,

Did you mean invariant NKT cells?

Could you give us more step-wise details of your protocol, as well as your gating strategy example (bivariates starting from Ungated cells down through to your target gate)?

I will say: I recommend titering even Fluidigm antibodies under your experimental conditions. I did a study about a year ago, using only Fluidigm antibodies. Of the 20 antibodies included in the panel, I used 6 at 0.5uL/test, 12 at 1uL/test, and 2 at 1.25uL/test. So, under your particular assay conditions, the recommended Fluidigm titer may not be optimal.


Mike
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cguidos

Contributor

Posts: 28

Joined: Tue Nov 18, 2014 3:10 am

Post Thu Oct 13, 2016 4:11 pm

Re: invariant NK cells

Hi James

We have also had difficulty finding iNKT cells in PBMC. I'm told that in contrast to mice, iNKT do not circulate much in human and so very few are found in PBMC. I'm also told that they do not survive freeze/thaw well so you might try fresh PBMC if you haven't done so yet

Cindy
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PSubrahmanyam

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Posts: 3

Joined: Fri Jan 16, 2015 7:15 pm

Post Thu Oct 13, 2016 5:18 pm

Re: invariant NK cells

Hi James,
The iNKT cell population in humans can be highly variable. Some individuals have extremely low (almost not detectable) circulating iNKT cells. So I would recommend testing multiple donors before you draw any conclusion.
Another thing you can do is to compare Va24Ja18+ population with CD3+CD56+ population and see if the numbers are close. You can also determine expected iNKT cell frequencies by trying the anti-Vb11 antibody on the same donors either on CyTOF (or flow).

Good luck!
-priyanka

P.S.>>I'm pretty sure you're already doing this, but just thought I'd mention that gating should be done on total CD3+ since many iNKT cells will be CD4 and CD8 double negative.
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jamesaries

Participant

Posts: 14

Joined: Thu Sep 22, 2016 2:49 pm

Post Fri Oct 14, 2016 12:06 pm

Re: invariant NK cells

Hi all,

Thanks for all the suggestions. I'll look at CD3+ CD56+ too.

Mike - yes I mean iNKT cells (sorry). Here's my gating strategy and iNKT cell 'positive' population (i.e. CD3+) & 'negative' population (CD19+) - as a PPT file.

James
Attachments
cytofFORUMiNKT.pptx
(225.45 KiB) Downloaded 401 times
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Oct 14, 2016 3:48 pm

Re: invariant NK cells

Hi James,

Thanks for the examples.

Could you give us more details about your staining procedure? Specifically: you say that your protocol "combines the cell surface followed by the nuclear staining protocol" (Fluidigm's Maxpar® Nuclear Antigen Staining Protocol?). I assume this means that you do the surface staining, then do the nuclear staining buffer step (which must contain fixative?).

If so, the Protocol says "This protocol is optimized for detection of antigens located within the nucleus; however the staining intensity of some surface markers may be diminished as a result of the fixation and permeabilization. Other protocols may enable adequate, although less optimal, detection of nuclear antigens, but allow for better preservation of surface staining."

In other words: you might want to do a test on a regular live cell surface staining protocol that uses eBioscience saponin perm, to make sure that your staining conditions aren't messing up your surface staining. I know we've had problems with stuff like this affecting some surface stains.


Mike
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gallia

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Posts: 1

Joined: Fri Jan 22, 2016 8:45 am

Post Mon Oct 17, 2016 9:25 am

Re: invariant NK cells

Hi James,

I have ordered Fluidigm's Va24Ja18+ also for staining of PBMCs. I will receive the antibody soon and perform my staining on from frozen samples too. Will keep you informed on the outcome.

Gaëlle

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