CyTOForum

Hi all,

We are planning to start a new project about histone modifications in osteoarthritis, and given that we can only use around 1M cells per hip for this project, we are trying using the Curiox Laminar Wash as an alternative to centrifugation to increase cell recovery.

The thing is, chondrocytes tend to stick to each other and form a matrix very quickly, therefore we were worried that it might be an issue because the LW relies in letting the cells settle for some time... our Curiox FAS recommended adding to 10% BSA to the well, incubate 30-60 min and subsequently remove the BSA before continuing with the staining protocol.

Now, we did a first experiment, with C28 cells, using one BSA coated well and one Non BSA coated well. We started with 1M cells for each condition and did an intracellular staining, using 1% SDS for 2 min for cell permeabilization.

The issue I have now is that looking at a (very basic) plot we made of the data we see that for all markers the staining of the No BSA sample is much higher than for the BSA coated, even tho we got a better cell recovery for the coated, and more events recorded.

I guess my question is, can this BSA coating be interfering with the permeabilization or the staining of the cells?

I am uploading the graphs we did in Cytobank. We stained for H4-116Cd, H4K20me1-143Nd, H3-176Yb, H3K27me3-161Dy and H3K9ac-166Er.

I would really appreciate any guidance in this

Isabel

Hi Isabel,

I think the first question is: how does this compare with the staining in your previous experiments *not* using the Curiox plates (ie, using regular 96 well plates or tubes)?

Put differently: what makes you sure that the higher no-BSA signal is "correct", and the "low" BSA signal is "wrong"? The no-BSA signal could be nonspecifically high, due to Abs sticking to the wells.


How did these samples look when they were running? Did both of them have the same background/streaking (at the same cell dilution), or was one of them higher? You can even screen for streaking by looking at the signal in these channels on the EQ beads (which don't have 116Cd or 143Nd): if there was a streak or other kind of unbound background during the run, the EQ beads events would have that.

For standard surface markers, normally you would look at background of a T cell marker on something like an NK or B cell that doesn't express it. Unfortunately, it's tricky to look at a lot of histone marks by cell population, as many/most of them are expressed on most of the major cell populations at one level or another.....


Also, I have to say, I've never heard of 1% SDS for 2min as a perm before: most protocols use methanol or some kind of saponin for longer than that....


Mike