Fri Feb 07, 2014 2:24 pm by robs
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.