Accuri benchtop c6 cytometer

Single cell suspensions were generated from whole prostate tissues combining all prostate lobes (as above in bacterial colonization) and iliac lymph node tissues by homogenization and filtration. Cells were washed in FACS buffer (2% FCS (Hyclone) in PBS (Gibco)) and staining performed using conjugated antibodies: PerCp-CD4, Alexa-488-CD4, and PE-IL17A (Biolegend), and PC-CD11b, FITC-CD273, PE-CD274, CD3, CD8, and IL4 (eBioscience). Samples were run on an Accuri-benchtop-C6 cytometer and analyzed using FlowJo™. For all analyses, unless otherwise stated, samples were gated on cell populations based on lymphocyte or monocyte size, as assessed by SSC and FSC, followed by gating for CD11b, CD3, or CD4. Intracellular staining was performed using IC fixation buffer (eBioscience) and Permeabilization Buffer (eBioscience). Staining was performed for 1 hour at 25C followed by washing in FACS buffer.

Flow cytometry was performed on single cell suspensions using these mouse antibodies; PerCp-CD4, Alexa-488-CD4, Alexa-647-FoxP3, PE-IL17A, FITC-CD25, PE-PD1 (Biolegend), PE-FoxP3, PC-IL17, FITC-CD25, APC-CD11b, FITC-PDL2, PE-PDL1 (eBiosciences). Flow cytometry was run on an Accuri benchtop C6 cytometer and analysed using FlowJo™ software. For all analyses unless otherwise stated, samples were gated on lymphocyte populations based on size, as assessed by SSC and FSC, followed by gating for CD4 positivity. Intracellular staining was performed by fixation and permeabilization using eBioscience Fix-Perm Intracellular staining buffers (Cat. Num 8222-49 and 8333-56). Staining was performed for 1 hour at room temperature followed by washing in FACS buffer (2% FCS (Hyclone), in PBS (Gibco)) Analyses were performed using FlowJo™ and data statistically tested using GraphPad Prism™ software, with tests described in respective figure legends.