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New WB injector and cell acquisition solution

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It is fine to promote your company's reagents. Just make sure they are relevant to CyTOF, and do so in moderation and style :-)
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mleipold

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Location: Stanford HIMC, CA, USA

Post Tue Jul 10, 2018 4:02 pm

Re: New WB injector and cell acquisition solution

Hi Regina,

I know there are a few places that are testing out the new wide-bore (WB) injector + CAS as recommended recently by Fluidigm, rather than the previous narrow-bore (NB) + MilliQ.

Stanford HIMC did our own testing, using all 3 of our standard PBMC assays. Basic protocols can be found below.
surface phenotyping: http://www.bio-protocol.org/e1382
ICS: http://www.bio-protocol.org/e1370
phosphoflow: http://www.bio-protocol.org/e1496

You can find a PDF of our results here (file was too big to attach here)....let me know if you have trouble accessing it: https://drive.google.com/open?id=1LxvF5 ... IZUqbywInQ


We present most of the data in a similar fashion to how Fluidigm presented their data: Median and CV ratio of (WB+CAS)/(NB+MilliQ) for several populations.

In short: in our hands, for all 3 of our assays, the previous method (NB+MilliQ) was as good as, and often *better* than, the new method (WB+CAS). For the surface phenotyping, normalization (Fluidgm or Finck) made the ratios more comparable, but never flipped it.


Since the new WB+CAS was no better than the original NB+MilliQ *and* there is also a significant increase in daily instrument maintenance due to salt buildup from the CAS, the Stanford HIMC is not planning to implement this with our current protocols.


Note: it's entirely possible that there *are* protocols where this new protocol would make improvements, like Dariush saw. However, it's definitely *NOT* every case......I'd recommend that your site do your own testing (ideally for each assay) to see if there's a difference.


Mike
Last edited by mleipold on Wed Jul 18, 2018 11:39 pm, edited 1 time in total.
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AdeebR

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Location: NYC

Post Tue Jul 17, 2018 11:36 pm

Re: New WB injector and cell acquisition solution

Hi Mike,

Thanks for sharing this. As you know, we've also been doing a lot of testing of the WB vs NB injectors, and in our hands, the WB+CAS provided a clear improvement in cell signal CV and population resolution over the NB+H20 configuration on all 8 Helios instruments that we've tested. Surprisingly, also includes the prestained cells that we shipped to Stanford and Natalia ran on the Page Mill Helios, which is the same instrument that you ran your tests on. This makes me quite puzzled about our discrepant results. It suggests that the relative difference in performance between the WB and NB injectors is not just related to the instrument, but also to the specific sample or antibodies used.

Very confusing...

Adeeb
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC
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cguidos

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Post Wed Jul 18, 2018 1:00 am

Re: New WB injector and cell acquisition solution

Dear Mike

My group has also tested the WB vs NB injector on our 2 Helios instruments as well as the new cell acquisition solution. Our findings echo Adeeb's - tighter CVs and thus better resolution.

Also, you may recall my prior post about the mysterious and sudden signal drop problem we were having intermittently but frequently on both instruments, but only in certain channels. Since we switched to WB+CAS we have not had this problem at all - huge improvement for us!

Cindy
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mleipold

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Post Wed Jul 18, 2018 4:45 pm

Re: New WB injector and cell acquisition solution

Hi Adeeb and CIndy,

It's definitely weird. I know of some other sites who are also doing their own NB+MilliQ vs WB+CAS, and are getting mixed results: most of the ones I'm aware of are getting results closer to the HIMC's than to Fluidigm's.


Adeeb: yes, Natalia ran your pre-stained sample on the Page Mill Helios, which is the same instrument for all the HIMC in-house testing sets. In fact, she ran your pre-stained sample on the same day as the ICS samples (Experiment #2: 6/27/18).

I took your sample's data out of the presentation, as I wasn't sure how widely to share that info yet (especially since we hadn't had the conference call about it yet). Yes, she did find that your pre-stained sample had improved CVs in some markers. However, she also had a ton of debris: I think she had only 20-25% recovery of LiveIntactSinglets from Ungated, compared with all the other HIMC samples at our more usual 50-60% recovery. So, in this particular case, if there was a sample stability issue, I'm not sure it's the best comparison.

Or, minimally, there's a strong sample-dependence involved. One difference between the HIMC protocols and many of the other protocols (such as Adeeb's CIMAC for the pre-stained samples, or Sheila's recent post back on the original "Strange signal shift" Cytoforum thread: viewtopic.php?f=4&t=582): none of the HIMC protocols use commercial Fix/Perms like Fluidigm Fix/Perm or BD....we use PFA freshly diluted to 2% or 4% (not 1.6%), eBio saponin perm, and methanol.

The only small exception is when we use Fluidigm barcode reagents: we use the Barcode Perm, but do *not* use the Fix 1 buffer. But, frankly, Barcode Perm is probably effectively the eBio perm.

In short, here's Natalia's current phospho workflow:
1. Thaw
2. Rest
3. Stim
4. 2% PFA to fix/stop. Although she has started doing 1.8% PFA, it helps with CD127 staining. (freshly diluted from 16%)
5. Fluidigm BC, including Barcode Perm buffer
6. Combine
7. Surface stain
8. Fix with 4% PFA (freshly diluted from 16%)
9. Methanol perm
10. Intracellular stain
11. Ir + 2% PFA (freshly diluted from 16%)
12. CyFACS wash, then 2 MilliQ washes

In the case of Whole-blood phospho, step 4 has smart tube stabilizer instead of PFA.

CyFACS = PBS + EDTA + BSA


Though, to be honest, insufficient fixation also doesn't seem to fully explain this: when I've had insufficiently fixed cells (old PFA, pipetting error so one sample doesn't get enough PFA, etc), generally *ALL* (or almost all) the markers are affected, often including Iridium. Additionally, the background goes up in most/all channels as the cells disintegrate and basically become a few intact cells mixed with free-floating debris. For insufficient fixation, it's not at *all* clear to me how only *some* markers would be affected, while others would be perfectly fine. Unless there's some weird middle ground where the cells aren't busting *quite* as fast, and there are some markers that are released more easily than others? My understanding is that CAS is an ionic buffer, which should reduce the osmotic stress on the cells, at least relative to MilliQ.


Since Natalia was the one who actually ran your pre-stained sample, hopefully she'll chime in to this discussion to clarify further.


Mike
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CRStevens

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Post Thu Jul 19, 2018 1:54 pm

Re: New WB injector and cell acquisition solution

Thank you Mike for your information.

I assume that you also had to run your cells slower with the CAS and WB injector as well? And most importantly, did anyone actually see a better yield from their sample efficiency in the machine? The higher ionic strength was supposed to stop our cells from popping or disintegrating in the water. If no one sees an increase in efficiency, then I really would question the reasons why it is worth switching.

* To clarify I mean cell yield on the machine, so if I put a million cells in how many am I actually recording as events back?

-Chad
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mleipold

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Location: Stanford HIMC, CA, USA

Post Thu Jul 19, 2018 2:51 pm

Re: New WB injector and cell acquisition solution

Hi Chad,

As far as I recall, we ran the CAS+WB HIMC samples at the same concentration (as measured by Biorad TC20) as we did the MilliQ+NB HIMC samples. It at least wasn't significantly different....no increased dilution factor. That said, the HIMC typically runs samples at ~250 events/sec anyway (around 0.8M/mL on TC20), which is lower than a lot of people run.

We also didn't see any notable increase in cell transmission efficiency/yield between the sets of samples.

I will say: with the WB injector (with or without CAS), your Event Length *does* get longer....back to the ~30 EL from v1 and v2 instruments, compared to the ~20 EL we've been used to with the (old NB) Helios. So, yes, if you're running at a higher concentration, you may need to dilute your sample further to maintain an acceptably small number of Doublets.


Mike
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mleipold

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Post Wed Jul 25, 2018 11:15 pm

Re: New WB injector and cell acquisition solution

Hi Chad, Adeeb, and Cindy,

Fluidigm of course won't state what is in CAS. You can make certain assumptions: the injector gets a lot of buildup, but the skimmer-reducer doesn't get that dirty. This implies that it's something that can completely burn up in the plasma, unlike the NaCl in PBS.

Doing some more research into mass spec-friendly buffers, it appears that ammonium acetate decomposes to gases at around 150C, and ammonium bicarbonate decomposes to gases around 40C. Since the spray chamber is at 200C, either of those would still give you a decent temperature margin for decomposition to hopefully prevent salt buildup.

https://doi.org/10.1016/S0040-6031(01)00585-8
https://en.wikipedia.org/wiki/Ammonium_bicarbonate
http://belhim.com/en/catalog/salt/ammonium-acetate/


Analytical Chemistry paper comparing Ammonium acetate with ammonium bicarbonate in the context of protein mass spec: http://dx.doi.org/10.1021/ac401020s

Note: they comment about how ammonium bicarb can foam a bit with heating, as the gas volatilizes quickly. I don't think this would be a concern for CyTOF, as we're still dealing with cells rather than free protein molecules in solution. Also, the buffer concentrations in that paper are usually quite high: 0.1M (100mM) and up. In fact, if you look at Fig 2, top row, 0.01M (10mM) doesn't seem to cause the same issues as the higher concentrations. I don't know what buffer/salt conc CAS has, but if it's just there as osmotic stress relief compared to MilliQ, 10mM might be enough.....

Otherwise, ammonium acetate should work too, just a smaller variance between decomposition temperature and spray chamber temperature.


- Note: I haven't tried running either buffer into my Helios(es), nor have I tried it with cells. I'm pushing Fluidigm to do this testing.....functionally, running EQ beads for 4-5 hours in each buffer (10-20mM) and seeing how dirty the instrument gets would be a good, easy place to start. The massive increase in instrument maintenance is one of the biggest strikes the new protocol has.

Also, if there's no buildup, then no need for the WB injector........


Mike
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mleipold

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Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

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Post Wed Aug 01, 2018 3:37 pm

Re: New WB injector and cell acquisition solution

Hi all,

Someone pointed out in an email to me that some information on the new CAS can be found in the SDS for the CAS from Fluidigm's website:

https://www.fluidigm.com/reagents/prote ... tion-100ml

Down at the bottom, click on "SDS" and then you can download the PDF.

In short: "Chemical family Mixture; contains ammonium nitrate". Although it doesn't give a concentration, pH, etc, it's still good information.


As discussed earlier, the buildup on the injector but not on the cones argues that it's a buffer that would burn completely, but it not volatile/able to decompose at ~200C from the spray chamber.

Ammonium nitrate would satisfy those parameters: ammonium nitrate doesn't melt until 170C, with a decomposition temperature around 210C: https://en.wikipedia.org/wiki/Ammonium_nitrate


Mike
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FlowjoVA

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Post Wed Aug 01, 2018 4:22 pm

Re: New WB injector and cell acquisition solution

According to the MSDS the CAS contains <0.1% of ammonium nitrate. I suppose one could start testing there and see if they could come up with what the optimum concentration would be. ;)
Joanne
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mleipold

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Post Wed Aug 01, 2018 4:29 pm

Re: New WB injector and cell acquisition solution

Thanks Joanne, good point.

Assuming <0.1% would mean w/v:
<0.1g NH4NO3/100mL @ 80.04 g/mol = <12.49 mM NH4NO3
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