Anti cd64

To assess bacterial infection in macrophages, HMDMs were harvested after adding non-enzymatic cell dissociation solution (Sigma-Aldrich, Oakville, ON, Canada) for 30 mins at room temperature. Cells were washed with PBS and prepared in FACS tubes. For surface staining, we used the following specific monoclonal antibodies provided from BD Biosciences for phenotypic analysis of human HMDMs: anti-CD3 (BV605), anti-CD14 (BV650), anti-CD80 (PE Cy7), anti-CD209 (PE) and anti-CD64 (PE). Finally, the viability marker 7-aminoactinomycin D or 7-AAD (ThermoFisher Scientific, St. Laurent, QC, Canada) was used to exclude dead cells from analyses. Afterwards, cells were fixed with 4% paraformaldehyde solution and then washed. For data analysis, samples were analyzed by flow cytometry with a BD LSRII Fortessa flow cytometer and DIVA software (BD). Viable gated cell singlets were analyzed for each sample and the percentages of mCherry^high CFT073 containing CD14⁺ HMDMs were determined. For Imaging flow cytometry, the same procedures were applied while cells were prepared in Eppendorf tubes. For surface staining, we used CD14-V450 and CD80-APC H7 for phenotypic gating of HMDMs. Samples were acquired using the Image Stream X MKII flow cytometer and analyzed with IDEAS software (Amnis) and representative images of singlets CD14⁺ HMDMs were generated expressing GFP^high CFT073 bacterial cells.

For the surface marker staining, the cells were labeled with the following monoclonal antibodies: anti-CD14 (Immunostep), anti-CD11b, and anti-CD64 (BD) and HLA-DR (Immunostep). Matched isotype antibodies were used as negative controls. The cells were incubated in the dark for 30 min at 4°C. The data was analyzed by flow cytometry using a BD FACSCalibur flow cytometer (BD Biosciences). The data was analyzed with Cell Quest Pro software (BD Bioscience).