Icys research imaging cytometer

The optimal dilutions of the antibodies were determined by staining a titrated range of antibodies on SK-OV-3 and HCT-116 cells. Antibodies were diluted to 0.1, 1, and 10 μg/mL in PBS with 1% BSA (Sigma) and 1% heat- inactivated human AB serum. Fresh cells (1–5 × 10⁶/mL) were stained and washed, and bound antibodies were detected by staining with either FITC- or DyLight-649-conjugated antimouse antibodies as secondary antibody. Dylight-649 conjugated goat antimouse antibody at a 1:200 dilution. The fluorescence signal was detected by a three-laser FACSCANTO II flow cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar, Inc.). For LSC analysis, fresh stained cells were fixed with 2% paraformaldehyde (PFA) and cytospun on a glass slide. Peripheral blood mononuclear cells (PBMCs) were used as a negative biological staining control. The fluorescence signal was detected by LSC using a three-laser iCys^® Research Imaging Cytometer (Thorlabs), and the mean fluorescent intensity (MFI) was reported for each antibody.

LSC was performed using a 3-laser iCys^® Research Imaging Cytometer (ThorLabs). Quantitative image analysis was performed using iCys 3.4 software. Individual cells were identified by contouring the DAPI-stained nuclei. CTCs were defined as cells that were DAPI⁺/CK⁺/CD45⁻. The cutoff for positivity of CK and CD45 was defined by isotype controlled irrelevant antibodies. The cutoff for FRα positivity was determined using the internal PBMC population in the CTC-enriched sample. Enumeration was determined for the classical CTC phenotype (DAPI⁺/CK⁺/CD45⁻) cells. The proportion of FRα⁺ cells among CTCs and PBMCs and the fluorescence intensity of FRα expression (expressed as MFI) were determined in the FRα⁺ cell population.