I have attempted to stain patient-derived cancer organoid cells that have been brought into suspension by accutase and trypsin treatment (for a normal split) for surface markers. I wanted to establish a staining panel for identifying cancer stem cells in the mixed population and analyze if they express some receptors of interest.

The panel I have chosen was tested for fluorophore compatibility on single-stained beads that could successfully be compensated with reasonable spillover values (none higher than 30).

We received organoid cells from a partnering facility freshly after a split, transferred them on ice (~10 min walking) and I proceeded directly with staining. Just for establishment purposes, I only had a full stained sample with a live-dead dye and 5 antibodies and a sample of the same cells stained just with the isotype controls. Now my issue is that some of my isotype antibodies show stronger signal compared to the actual targeting antibody after the compensation has been done.

What could cause this and how do I troubleshoot?