Sat Jan 13, 2018 4:22 am by GregBehbehani
Hi Beth and Cindy,
I would certainly agree with everyone else that this is quite weird. But I would suggest a few things that you may or may not have tried/thought of so far.
1. In my experience freezing always disrupts the cell membranes to some extent, and this would be expected to be most significant when a relatively gentle fixation is used (e.g. 1.6% PFA). I suspect the main difference between the frozen and never frozen samples is that the frozen samples are physically breaking apart is some relatively subtle way (loss of loosely attached surface proteins, increased intracellular/intranuclear permeability, decreased interaction between protein binding partners, etc.). If that were the case, this effect could vary based on protein, epitope and antibody clone so it may not seem to be very consistent. This could also lead to a signal and/or background decrease (due to antigen loss) or increase (due to increased antigen accessibility).
-Regardless of the cause, this should be fairly easy to sort out, as this effect would be dependent on the time the cells have been diluted in pure water. I would suggest repeating the experiment, but this time dilute both the frozen and never frozen cells in aliquots of pure water and let them sit at room temp for different periods of time (1m, 5m, 10m,....60m, etc.). If some sort of physical degradation is at play, the longer the cells sit diluted in pure water the greater the difference will be between frozen and never frozen samples (irrespective of whether or not they are running in the machine).
2. I'm really puzzled when you say "The signal drop happens with certain channels regardless of what marker is in them, happens with both human and mouse cells, and with both in-house and Fluidigm conjugates." Could you provide more information about this?
-Specifically, is EVERY effected antibody different when this happens, or just some are different? (I ask this, because you seem to be having a fair bit of M+1 spillover, so I think most of the TCF1_Er167 signal could actually just be spillover from the "bright" B220_Er166 signal. If that's the case, any "dim" antibody on Er167 would show the same pattern, but only if there is a "bright" antibody on Er166. You might have already checked for this. It also seems to be happening in the most sensitive part of the mass range, where spillovers might be most easily detected.
-Does this happen with pure metal in solution mode, or only with stained cells? If it doesn't happen in solution mode, I can't imagine any way this could be a detector, ion optics, or ventilation issue. If it happens in solution mode, it couldn't be a cell or antibody issue. This test should narrow things down quite a bit.
-What do you run just before your samples? Water, wash solution, or something else? It's hard to reduce the pH of a strong acid by diluting with pure water, so if you run sample too close to wash solution (even with water in between) it will strip the metal out of the chelator and give high background and low signal until the cells themselves buffer the acid away, which could take a while. It could also alter the antibody antigen interaction (again in a antigen and clone specific way). This could also be somewhat metal dependent, as different metals have different affinities for the chelator.
-Because of how the beads are made, they wouldn't show most of these effects (except for the portion of the M+1 spillover that's due strictly to mass tailing), which is potentially consistent with what you're observing.
Please do give us some follow-up. I'm incredibly curious to know what's happening.
best of luck,
Greg