Strange signal shift
I have encountered a strange phenomenon while recording data from a big barcoded experiment last week, and after hours of trying to figure out what is wrong I still did not come up with a likely explanation.
Here is a short description of my protocol:
I thawed cryopreserved PBMCs, stimulated for 5 hours with PMA+Ionomycin plus BrefeldinA/Monensin or BrefeldinA/Monensin only. After stimulation, I performed cisplatin viability staining, fixed the cells with 1.6% PFA, washed with CSM and left at 4C overnight.
The next day, I performed Palladium barcoding (0.02% Saponin, i.e. 1:50 dilution of eBioscience 10x Permeabilization buffer in PBS), pooled the samples, stained with surface markers, stained for Cytokines using 1x eBioscience Permeabilization buffer, and incubated with Ir intercalator in 2% PFA/PBS for 48 hours.
On the day of data collection, I resuspended the cells in 1 ml of normalization beads dilution and measured cell concentration. I collected data on a CyTOF2 using the supersampler in total 9 runs of about 1 hour each. Directly before each run, I diluted my initial cell/beads mixture about 10-fold to a concentration of about 500 000 cells/ml. At the end of the day, I concatenated all the files together.
What I noticed when I looked at the concatenated files was that for 4 of my channels (148Nd, 150Nd, 152Sm, 154Sm), the signal was shifting upwards during each of my 1-hour runs. Each time after I prepared fresh sample dilution, the signals came down for a short period of time before going up again. Other channels seem not to be affected (except maybe very weakly 149Sm, and 146Nd), the bead signal in all the bead channels (including 151 and 153 in the same mass range as the affected chanels) is steady as well. The affected markers are, except for IL-6 154 Sm, surface markers. I see this phenomenon independent of data processing (i.e. on the raw data, data normalized using the Matlab normalizer, on all my debarcoded files).
I was running a panel including the same surface markers, but stained for phosphorylated intracellular signaling molecules following methanol permeabilization, the day before and did not observe this signal shift. Another person was running samples directly after me and also did not observe something similar.
Before starting the experiment, I tested my cytokine panel (but without the barcoding step and using the loop system instead of the supersampler), and everything was fine then. (I only collected for 2x10 minutes then, so a shift over time might not have been that obvious for that short period of time, but still the signal seems to be very steady in this earlier experiment).
I am attaching a pdf to illustrate this problem. I'm showing plots of time vs. channels 147-155, for ungated files before normalization and debarcoding.
I would be happy for any explanation or suggestions of what tests to run in order to figure out what might be the problem here.
Thanks in advance,
Johanna