I need assistance quantifying migrated PBMCs using a transwell assay. PBMCs (50 000 cells in 50 µl) were seeded in the upper chamber of a Corning HTS Transwell 96-well permeable support plate (insert pore size 8 µm) in serum-free RPMI 1640 medium with GlutaMAX. The lower chamber contained medium supplemented with or without the chemoattractant, 10% FBS, and the plate was incubated for 24 h. After incubation, the non-migrated cells were removed from the upper chamber. The migrated cells that adhered to the underside of the inserts were fixed with 4% paraformaldehyde for 30 min and stained with 0.2% Crystal Violet for 10 minutes, and rinsed with distilled water. The stained PBMCs were imaged to assess the number of cells that migrated through the inserts. Since PBMCs are semi-adherent, the non-adherent cells migrated to the bottom of the wells, where images were also taken for quantification. While I can manually count the Crystal Violet-stained adherent cells in ImageJ using the cell counter, counting the non-adherent cells will be more challenging due to their more concentrated distribution at the edges of the wells (see image), especially in the chemoattractant group where more migration is expected. Any assistance on how to quantify this in a quick and easy way would be greatly appreciated! Please note that some cells do adhere to the bottom of the wells, making it time-consuming to harvest each well of a 96-well plate and count the cell suspension using a Countess. I have also tried Crystal Violet staining in the bottom wells to measure the absorbance with a plate reader, but the results are inconsistent, as some cells are washed away during the process.