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Yield of cells in CyTOF

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ido

Participant

Posts: 1

Joined: Tue Nov 12, 2013 11:55 am

Post Thu Feb 06, 2014 11:36 am

Yield of cells in CyTOF

In all the experiments and runs on the device I get about 10-15 percent of the cells from which I started staining.
At the beginning of staining I start with 1.5 to 2 million cells per sample.
Did anyone get a different cell output? What protocol do you use?

Ido
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Feb 06, 2014 8:03 pm

Re: Yield of cells in CyTOF

Hi Ido,

There are a couple things to address:

1. Because there are more staining steps in CyTOF than in many flow experiments, you generally have more wash steps. Therefore, you will lose more cells as you go along.

2. Fixation is ultra-important: we do not recommend using a stock of 16% PFA that is more than 1 month old. Do *NOT* assume that just because your PFA is fine for a standard flow experiment that it is fine for CyTOF: since flow samples usually remain in PBS or FACS buffer, they do not undergo the same osmotic stresses as CyTOF samples washed with MilliQ water and then resuspended in MilliQ water. A typical way to tell if this is affecting your samples is to watch your pellets at the end of the protocol:

If you finish steps in PBS/CyFACS and start doing MilliQ water washes, and then start noticing your pellet getting smaller and smaller, you're probably not sufficiently fixed. Additionally, there will be more background in your samples because of increasing debris.


3. Most importantly: the CyTOF machine itself has a cell transmission efficiency of only 20-30%. In other words, if you give it 1 million cells, the MAX you can usually expect to recover would be 200-300K cells as "Live Intact Singlets" (not "Found Cells", which includes debris and other background) . This is an inherent design issue to the CyTOF: cells stick to the sample loop, nebulizer tubing, nebulizer, and are lost to the spray chamber. This occurs even if you have ultra-clean nebulizer tubing and nebulizer, and the machine is tuned properly.


-- So, If you plate 2 million viable cells, you shouldn't expect to recover more than ~300K "Live Intact Singlets" after wash loss (#1) and machine loss (#3), even if your PFA is good (#2).


-Mike
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robs

Contributor

Posts: 40

Joined: Mon Dec 02, 2013 3:42 pm

Location: University of Connecticut

Post Fri Feb 07, 2014 2:24 pm

Re: Yield of cells in CyTOF

Hi Ido,

Unfortunately a lot of cell loss is expected, as Mike explained.

We made a few modifications to the MaxPar staining protocol to recover about 25-20% of starting cells in a PBMC sample, I'll give you the specifics. Sorry this turned into a long response.

We cut out a number of the wash steps which were a major factor in the cell loss (we counted after each step). We only do a single wash after antibody staining and if we are doing viability staining with Rh-intercalator or Cis-platin we perform it at the same time as the surface stain to remove a wash step.

Intracellular staining is done at the same time as Ir-intercalator staining, removing another wash step.

Finally we only perform 2 buffer washes and 1 water wash before running on the instrument, the MaxPar protocol mentions 4 total washes.

Additionally, we leave our cells pelleted in Ba-free PBS until right before we plan on running them, then we do the final water wash. We noticed after an hour or two even the fixed cells were beginning to swell/break apart. We have left fixed cells overnight in PBS without too much loss.

On our CyTOF 2 we only lose about 60% of the cells in the instrument, but we are mostly running PBMC so they are less likely to clump and get stuck in the tubing than a cell line or other tissue.

We developed these modifications after talking with other users and with DVS, these protocol adjustments are pretty typical and people usually see improved cell recovery.

Hope this helps,

Rob



Notes about the staining protocol:

If you are using the Rh-intercalator for viability you need to do the staining at 37 degrees for 20-40 mins before you add surface antibodies and incubate at 4 degrees. Rh seems to need the higher temp when used for viability.

If you are doing cis-platin for viability, you can add it during the last minute of your surface antibody incubation, it works fine at 4 degrees, as does the Ir-intercalator. Just make sure to wash out the cis-platin right away.

It seems that for good staining, the Ir-intercalator should be given at least 30 minutes, we sometimes use slightly higher Ir concentrations (30%) to make sure we identify all our cells

Keep in mind if you combine steps like this you still need to use a small staining volume for antibody binding so the amounts of cis-platin or DNA intercalator you add will need to be much less so you don't overstain.

Also, some buffers have a lot of metal background (usually Ba) so for some staining we have to add more PBS wash steps before we run otherwise our background is too high.

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