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CD4+ Yield in Whole Blood

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CRStevens

Master

Posts: 60

Joined: Thu Jul 17, 2014 5:07 pm

Post Wed Jun 24, 2015 8:06 pm

CD4+ Yield in Whole Blood

Hi,

I recently have been going back to a lot of our data and realized that my CD4+ yields are really low compared to what we should be getting. I understand we lose a lot of cells in the washing and in the introduction into the machine, but on average I'm getting about 10,000 CD4+ cells from 1mL of fresh whole blood. Do other people get about the same? If so has anyone figured out a way to increase that yield? My percentage of CD4+ is in a normal range, so it's not that I'm losing CD4+ cells specifically, just that I'm getting really bad efficiency (or maybe it's average?).

-Chad
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ndawson

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Posts: 13

Joined: Wed Jan 08, 2014 4:40 pm

Post Wed Jun 24, 2015 8:40 pm

Re: CD4+ Yield in Whole Blood

Hi Chad,

That is super low CD4+ numbers for 1mL of whole blood. We've looked at CD4 cells on ~70-100uL of whole blood (human) and got 30,000-50,000 CD4 cells. Could you maybe share your protocol so that we have a better idea of hwo you are processing your cells?

Nick
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CRStevens

Master

Posts: 60

Joined: Thu Jul 17, 2014 5:07 pm

Post Wed Jun 24, 2015 8:46 pm

Re: CD4+ Yield in Whole Blood

Thanks Nick,

We basically use the procedure on the BD FACS/Lyse protocol:
stain cell with Ab cocktail for ~30 minutes
Add 1:10 of 1x BD FACS/Lyse for 10 min at RT (pre-warmed at 37C)
wash with PBS twice
Add 500ul of BD Fix/Perm buffer with Ir intercalator overnight
wash twice with PBS, then once with water
resuspend in 1:10 EQ4 bead in MiliQ water, the run.
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mkunicki

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Posts: 17

Joined: Wed Apr 15, 2015 8:43 pm

Post Wed Jun 24, 2015 9:04 pm

Re: CD4+ Yield in Whole Blood

Could you show us your gating strategy for Intact cells also? That might be why you're seeing low CD4 specifically, but if you meant you have low yields in general I'd suspect it's a matter of your single cell suspension.
Also, what cell concentration do you dilute to with 4EQ beads, just before pushing your sample into the CyTOF sample loop?

Best,
Matthew
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Jun 24, 2015 9:17 pm

Re: CD4+ Yield in Whole Blood

Hi Chad,

Have you monitored the size of your cell pellet throughout the procedure?

In other words, how big is it at the end of your procedure vs how big it was after the FACS/Lyse, and after the first PBS wash after the Ir?

What RCF (xg) are you using for your spins?


This could help you figure out whether there is a particular step where you're losing most of your cells.


Mike
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billog

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Joined: Wed Sep 17, 2014 10:53 pm

Post Wed Jun 24, 2015 9:25 pm

Re: CD4+ Yield in Whole Blood

You should never need to process more than 500uls of whole blood to get good T cell #'s. I've used the BD lyse/fix with success (10mls of !x diluted lyse/fix for every 500uls of WB) but we have also had success with WB lyse only BD protocols

Are you counting cells before you inject them?

Sounds to me like your cells are popping when you resuspend them in 100% ddH20 and this is most likely because they are not sufficiently fixed. Make sure your fixative is not too old or has been compromised in some way...
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CRStevens

Master

Posts: 60

Joined: Thu Jul 17, 2014 5:07 pm

Post Thu Jun 25, 2015 1:55 pm

Re: CD4+ Yield in Whole Blood

Thank you to everyone who replied:

Matt,

What we're seeing is a general low yeild. It is not CD4+ specific. My runs are usually around 150-300 events/second.

Mike,

We have monitored the cell pellet before and found it to decrease, so we increased our spins after fixations to 800xg for 5 min. We also see that we do lose more after the water washes, which is why we've decreased the washes to 1 wash.

We do not cound them before we run, but maybe should try this.

As a second question do people do a "hard" fix after the fix/perm with the Ir? Does this help with the yield and how do people do this?

-Chad
Attachments
CyTOF backgating.tiff
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Jun 25, 2015 4:14 pm

Re: CD4+ Yield in Whole Blood

Hi Chad,

Do you happen to have a screenshot of what the acquisition window looks like as you run the sample?

Like Bill, I'm suspicious that you're not completely fixing the cells, and they're busting in the MilliQ. While MilliQ can cause cells to pellet differently than in PBS, the main source of loss in MilliQ is lysis.

Based on your TIFF, the Ir-bright population in your first gate is only ~25% of total Found events. This makes it likely that either you're not washing out the residual Ir, or you're lysing the cells. If you're lysing, then you should have background in just about every channel you measure. This will show up on the Acquisition screen as vertical streaks of varying density (from speckles to Sharpie-like tire tracks). This will also show up as background in all of your Events. This even includes background in all of your Bead events: take a look at the background levels in all of your Marker channels, in all of your Bead events.

Remember, a Found Cell Event is triggered when *any* of your channels satisfies the Event Length and Sigma parameters. This then grabs across *all* channels for the time-window of that particular cell event....if you have streaking, that time-section of the streak will be captured in the Found Cell data.


Mike
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CRStevens

Master

Posts: 60

Joined: Thu Jul 17, 2014 5:07 pm

Post Thu Jun 25, 2015 5:20 pm

Re: CD4+ Yield in Whole Blood

Hi Mike,

I don't have a screenshot of the acquisition, but here are some plots showing no background issues. I don't see the streaking that your talking about either. The acquisition looks pretty normal to what I've seen before from Fluidigm and others. I'm going to run a quick experiment and just look by flow to see where i'm losing my cells in the processing. I'm assuming that my loss from the machine will be the same either way?

Could you explain how you do your fixation? Do you do a hard fix (4% PFA) after your fix/perm Ir stain?

-Chad
Attachments
background.tiff
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Jun 25, 2015 5:38 pm

Re: CD4+ Yield in Whole Blood

Hi Chad,

I work primarily with PBMC, rather than WB. In either case, after surface staining (and then RBC lysis) and live-dead, I do 2% PFA overnight at 4C. Day 2, I use saponin as my perm, stain for Ir; I don't do a second PFA fix since I seldom do intracellular staining (if I do, then I do a second fix during Ir).

You can just try counting your cells at each step. While we use a Vicell during the initial thawing and counting, we usually use one of the Biorad cell counters before injecting into the CyTOF. Those only use 8-10uL of your undiluted sample per reading, so you're not using up much of your overall sample if you were to test as you go along.


Mike
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