Help! Unexplained Background
Lately I've been struggling with high background and low signal. Until recently my cells have been staining brightly with very little background, but in the last few experiments the intercalator-associated signal has dropped and background is higher than I've ever seen across all antibody channels. I have completely standardized pelleting and supernatant removal, so I'm relatively sure this isn't caused by inefficient washing steps. So I did the only thing I could think of... I had been using the same bottle of DVS buffers for many months now, so I decided to try making an in-house CSM (PBS+BSA+azide) and comparing that to my old bottle of DVS CSM in a small experiment. This brought my signal back up, but I'm still seeing background in certain channels containing antibody (mainly channels designated for high-expressing markers such as CD45 and CD3). Just to be thorough, I measured the pH (7.5) and tested for microbiological contamination (culturing on LB agar) in my old bottle of DVS CSM. As far as I could tell, nothing seemed out of order.
This is all very puzzling, and has opened the door to many more questions. Any help offered would be so greatly appreciated!
1) In general, what are the most common causes of background/streaking? What about uncommon ones?
2) What could cause a drop in signal that is concurrent with bright background across all antibody channels?
3) What are the most common sources of background that only occur in some channels - in my case, CD45-156Gd, CD11c-162Dy, HLA-DR-174Yb, CD3-175Lu, CD8-176Yb are the worst offenders. All of these have been titrated and have shown very little background in some previous experiments (though not reliably... see #6)
4) If there is Ba contamination, which channels would be affected?
5) If I'm only seeing background in my high-expressing markers, would that indicate that the problem isn't due to improperly titered antibodies (because high-expressing markers can bind up more antibody than low-expressing markers, therefore it is more likely that I would see excess antibody for low-expressors)?
6) I've noticed background seems to fluctuate a great deal between experiments and even between samples in the same experiment. I'm relatively sure this isn't due to technician error because I have spent a considerable amount of time standardizing my technique. What else could account for this?
7) My background seems to increase and decrease with cell concentration (even if resuspended in the same sample volume - for example, if I resuspend 1e6 vs 2e6 cells (stained with same concentrations of antibody) for acquisition in 1 mL of water, background tends to be higher in the 2e6 cell sample. Has anyone else noticed this to be true?
8) Recently I've been resusupending my pellets in residual volume by light vortexing (as indicated in MaxPar protocols). Could this be increasing my background due to more cell debris, etc?
9) Also recently I've also begun staining my cells on ice outside of the biological hood using non-sterile supplies (reagent basins, tips, etc.) to save money. Could this be contributing to metal contamination?