'towers' during acquisition in specific channels.
A question about a recent experiment and the unusual appearance of staining over time in CD138 -- wonder if anyone has seen similar and tracked down the cause? We stain CD138 using CD138-APC followed by APC-176Yb and found that in contrast to previous experiments, staining over time was non-random, with discrete clusters of positive cells in the first 2-3 tubes, and unexpectedly few positive cells in the remainder. The samples are murine bone marrow/spleen preparations, with number of events acquired in each tube from 80k to 497k (first 4 tubes: 497k, 256k, 104k, 77k)
A link to a .pdf illustrating the problem is as follows:
https://www.dropbox.com/s/oimrenr2nrorr ... 8.pdf?dl=0
The first slide is the intensity of CD138 staining (y-axis) in each of 14 tubes (1-14) run consecutively over time (x-axis). In tubes 1-3 in particular positive cells appear in patchy towers and thereafter hardly any positive cells are seen at all. We use barcoded mixed samples, mixing 4 samples in each tube, so feel it is unlikely that this is related to the staining of an individual tube.
The second slide is CD138 staining from our previous experiment using a very similar staining protocol. This is much more what we expected to see: a much more random distribution of CD138 positive cells over time with positive events in all tubes.
The third slide is another marker CD19 (149Sm) from the same experiment as the unusual staining again we are perhaps losing positive events over time and a few towers are noted in tube 2.
In other words, to summarise, it seems to me that we have far fewer CD138+ events than expected, and that the distribution of positive events over time is non-random. I've searched the forum as best I can and have not been able to find similar; our hypotheses are primarily ?too concentrated a sample or clumping (but filtered immediately prior to running) ?machine-related technical phenomenon. Any thoughts as to what might be causing this, and anything we should be thinking about prior to our next run in a month's time?
Many thanks, in advance,
Shaun.
(Visiting fellow, INSERM, Paris)