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"Homemade" Cytof running buffer

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EHaasDFCI

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Joined: Thu Jun 15, 2017 5:34 pm

Post Fri Apr 26, 2019 7:10 pm

"Homemade" Cytof running buffer

Hi Everyone,

Has anyone had any success in replicating a buffer similar in properties to CAS? We have been running more and more in CAS and think it is a good method, however the prices for such reagents seem to be skyrocketing. If I could make my own stock of a solution with similar properties (not trying to infringe on any IP here), that would be much better for our lab and resources. If anyone has any recipes or has had any luck with such a thing, I would love to hear from you!

Happy CyTOFing,

Eric
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mleipold

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Posts: 5671

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Apr 26, 2019 10:58 pm

Re: "Homemade" Cytof running buffer

Hi Eric,

Short answer: I don't know anyone making their own CAS. I can't find it at the moment, but there was an MSDS for the CAS that mentions ~0.1% ammonium nitrate. I think that came out to around 12mM.

There's probably more in CAS than that, though.


Mike
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mleipold

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Posts: 5671

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Apr 26, 2019 11:04 pm

Re: "Homemade" Cytof running buffer

Google "fluidigm cell acquisition solution msds" and you should be able to find "SAFETY DATA SHEET. Product Identifier: Maxpar® Cell Acquisition Solution. SDS ID: MSDS-201237. Catalog ID number: 201237."
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mleipold

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Posts: 5671

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Apr 29, 2019 7:26 pm

Re: "Homemade" Cytof running buffer

Hi Eric,

Assuming that CAS is indeed 12mM, you might be able to dilute it at least 2x with water. Assuming that the main effect of CAS is reducing osmotic shock to the cells (relative to 18MOhm MilliQ water), you could probably afford to dilute it at least a little bit (though I don't have osmotic stress calculations for cells dropped into pure water as the comparison).

Regarding alternative buffers. The CAS MSDS lists ammonium nitrate as (at least one of) the CAS component(s). I've been told by Fluidigm that they went with ammonium nitrate because it is completely dissociated in the argon plasma (ie, unlike PBS, you won't get white salt buildup on your cones).


Ammonium nitrate melts at ~170C and decomposes at ~210C: https://en.wikipedia.org/wiki/Ammonium_nitrate
- since the spray chamber is only 200C, it doesn't seem like this would be high enough to prevent buildup in the injector, which is why running CAS builds up in the injector.

***Specifically, I was wondering whether ammonium bicarbonate might substitute:
Ammonium bicarbonate: decomposes to gases around 40C: https://en.wikipedia.org/wiki/Ammonium_bicarbonate


I was also told that the main issue around carbon-containing buffers in ICP-MS is the carbon buildup under the current plasma conditions (ie, without increased oxygen to aid in combustion). He said that assuming 1M cells and an approximation of cells as 10um cubes, you would get about 1mg of carbon (eg, 1mg carbon/1M cells).

**Assuming:
1 cell = 10um cube (10um = 10e-4 cm; cubed = 1e3 x 1e-12cm3 = 1e-9cm3)
1cm3=1mL; therefore 1e-9 mL per cell
Density of cell ~ density of water (1g/cm3; 1 g/mL; 1000mg/mL)
- above gives you 1e-6 mg/cell
Therefore 1M cells would give you 1mg/1M cells.
- perhaps a bit lower: https://en.wikipedia.org/wiki/Compositi ... ition_list
- at ~0.2 mass fraction Carbon in the human body, that would give you ~0.2mg carbon/1M cells


However, according to my calculations below, a 12mM solution of ammonium bicarbonate (NH4HCO3) used to dilute cells to 0.8M/mL (our standard running concentration; approx 200 events/sec) would give approximately the same amount of carbon in ~7hr as from the cells:


**If my math is correct, assuming:
12mM ammonium bicarbonate (MW 79.056 g/mol; NH4HCO3);
- I chose 12mM bc that's an approximate ammonium nitrate concentration from the "<0.1%" ammonium nitrate on the CAS MSDS....could be lower.
Helios PSI flow rate: 30uL/min (1.8mL/hr)
sample dilution to 0.8M cells/mL suspension (our standard running concentration for PBMCs; corresponds to about 200 events/sec)

-then in 12mL of cell suspension (roughly 7hr runtime, or roughly 1 day of runtime), you would get 11.38 mg of ammonium bicarbonate. ~12 mg carbon/79 mg NH4HCO3 = 0.15 mass fraction carbon --> ~1.7mg carbon from the buffer


-compared to: 12mL cell suspension: 0.8M cells/mL *12mL * 0.2mg carbon/1M cells= ~1.9mg carbon from the cells


** So, according to the above, 0.8M cells/mL should give you roughly equal split in carbon coming from the cells and from the NH4HCO3 buffer, giving an effective *total* (buffer+cell) carbon content comparable to ~400 events/sec.


According to p. 108 of the Helios manual:
Average event rate ~500 events/sec
Peak throughput ~2000 events/sec


** As I understand it, the above average event rate is primarily limited by the resolution/overlap of the ion clouds and clogging propensity, moreso than what the cones can handle in terms of elemental composition (carbon content). Therefore, I'm not sure why this would be an issue. And that's even assuming we used 12mM ammonium bicarbonate; 1-5mM might be enough to reduce the osmotic stress on the cells.



I can also confirm that ammonium bicarbonate solution bubbles when immersed in even hot tap water, so the decomposition information above seems plausible.

It's not something that we have tested yet: we haven't seen the tailing issue nearly as much as some other labs and instruments (and as seen in Fig 6A of the recent Lee at al multisite paper: https://doi.org/10.1101/600130 ), so this has been a low priority for us.


** If anyone tries out alternative buffers, please post and let everyone else know.....it's been a major topic of discussion for the last year!


Mike

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