Facsaria sorp instrument

Blood was collected prior to inoculation to establish baseline values, then at each time point outlined above (Figure S1). The percentage of cells expressing CD4, CD8 and CD21 surface antigens was determined by incubating 50 µL of EDTA-treated blood with mouse monoclonal antibodies directed to the feline markers CD4 (Fisher, clone 3-4F4, FITC), CD8 (Southern Biotech, clone fCD8, PE), and CD21 (Bio-Rad, CA2.1D6, AF647) at the manufacturer’s recommended volume per test for 20 min in the dark at 4 °C. Red blood cells were lysed, and samples fixed using the TQ-Prep workstation and IMMUNOPREP reagents (Beckman Coulter Inc., Brea, CA, USA). Unstained and single stained controls were prepared for each experiment. Data were acquired using BD FACSDiva™ Software (Diva 9.0.1., San Jose, CA, USA) interfaced with a BD FACSAria™ SORP instrument (Becton Dickinson, San Jose, CA, USA). Data were analyzed using FlowJo 10.8.0. (Ashland, OR, USA). Compensation values were determined using single stained controls. Gating proceeded from singlets to lymphocytes to CD4, CD8, or CD21 markers. The percentage of lymphocytes positive for each marker was evaluated over time and compared to baseline values (0 dpi) and naïve control data to compare alterations in lymphocyte immunophenotype in response to SARS-CoV-2 infection.

Flow cytometry analysis of circulating lymphocyte lineages was performed as previously described [41 (link)]. Briefly, 50 μL of EDTA-treated blood was treated with mouse monoclonal antibodies targeting CD4, CD8 and CD21 feline cell markers (Fisher, clone 3-4F4, FITC; Southern Biotech, clone fCD8, PE; and Bio-Rad, CA2.1D6, AF647 respectively) to determine the percentage of each cell type. Unstained and single stained samples were used as controls. BD FACSAria™ SORP instrument (Becton Dickinson, San Jose, CA, USA) containing BD FACSDiva™ Software (Diva 9.0.1., San Jose, CA, USA) was used to obtain data, and were analyzed using FlowJo 10.8.0. (Ashland, OR, USA).

PC3 and RWPE-1 cells (at approx. 50% confluence) were detached using trypsin then consecutively washed with 10% FBS-medium and PBS. Detached cells were stained for flow cytometry in PBS according to antibody manufacturers’ protocols (15 min at +4 °C, in the dark). Antibody details were as follows: anti-CD44-BV421 (clone BJ10, BioLegend), anti-CD90-BV510 (clone 5E10, BioLegend), anti-CD133/1-PE (clone AC133, Miltenyi Biotec), anti-CD57-PerCp-Cy5.5 (clone HNK-1, BioLegend), anti-CD24-APC-H7 (clone ML5, BD). Cells were washed with PBS and analyzed using a 6-laser FACSAria SORP instrument (BD Biosciences, USA).
Median fluorescence intensity (MFI) values were obtained from FACSDiva 6.1.3 software for each surface marker under study in the overall PC3 and RWPE-1 cell populations. The overall cell populations were gated based on light scatter properties and median signal intensities were exported for them. Immunophenotypes of the model cell lines were monitored several times throughout cell passaging. Average MFIs and their SDs were calculated for surface markers from different passage data.