Distinguishing CD45+ Cells and CTCs via Multimarker Immunofluorescence

To differentiate CD45⁺ cells and CTCs in our sample, we performed a live cell stain using direct immunofluorescence (DIF) with an antibody cocktail (Supplementary Table S1). The cocktail included 1 uL of PRAME [30 (link)], MCAM [31 (link)], MCSP [32 (link)], c-Kit [33 (link),34 (link)], PD-L1 [4 (link),5 (link)], MART-1 [35 (link)], and CD45, and 0.5 uL of hoechst nuclear stain (20 mM). FC block (BD Bioscience, Franklin Lakes, NJ, USA) was added to the cells at a concentration of 1 µL FC block per 100 µL of blocking solution, and incubated for 15 min at 4 °C. Next, 1 µL of each antibody from Supplementary Table S1 and Hoechst stain (1 ug/mL) were added. Cells were incubated at 4 °C for 20 min in the dark. The cells were washed with 1 mL of staining buffer (0.5% BSA, 0.02% NaN₃ in PBS) and spun at 300 g for 5 min. The cells were washed again with 1 mL of staining buffer and then centrifuged at 300 g for 5 min. Staining buffer was gently aspirated off, and the cells were resuspended in 200 µL PBS and plated on the CytoSort array, prepped with Corning Cell-Tak (final array volume 2.2 mL).