Hello All, I am currently optimising an SOP for cytospin using cell lines but am observing some aggregates on my slides (after cytospin) that I want to avoid.

I use disposable cytofunnels, reusable metal clips and poly-l-lysine coated slides. My current protocol involves loading my cell suspension into the funnel, cytospinning (800 RPM x 3 min), drawing a hydrophobic barrier around the 'cell spot', fixing with 4% PFA for 10 mins and doing 2 washes with PBS. Unfortunately, when I image the slides after, I notice debris/aggregates on the slide. I don't think these are dead cells but artifacts as they have very weird shapes (please see image below).

I know that this debris is not from the cytospin (gave it a thorough clean before using), or the fixative (aggregates were present before adding fixative) or from the cells (aggregates were present when I just cytospun PBS with no cells).

I will be using the cytopsin for patient samples and identifying rare cells so want to minimize these artifacts as much as possible. I just wanted to ask if anyone else has experienced similar issues and how to avoid?