Flojo software

To determine T cell surface markers for suppressor phenotypes, T cells were labeled in flow cytometry buffer (FCB; 1× PBS buffer containing 2 mm EDTA and 0.5% BSA) with fluorochrome-conjugated anti-human CD3, CD4, CD8, CD27, and/or CD28 (Biolegend and BD Biosciences) for 30 min on ice in the dark. These cells were then fixed in 1% paraformaldehyde (PFA) overnight at 4 °C, washed and resuspended in FCB for flow cytometry analysis of these surface markers. To determine IFN-γ and Ki67 intracellular expression, T cells were first labeled with the surface fluorchrome-conjugated antibodies, CD3, CD4, and CD8 (BD Biosciences), and then permeabilized based upon Cytofix/Cytoperm (BD Biosciences) manufacturer instructions and then labeled with fluorochrome-conjugated anti-human IFN-γ and Ki67 (BD Biosciences). To assess surface and intracellular staining of antibodies samples were processed on a BD LSRFortessa or BD LSRFortessa X-20 (BD Biosciences) and analyzed with BD FACSDiva and FloJo software (FlowJo, LLC). Ten thousand to thirty thousand cell events were recorded, and dead cells and debris were excluded based upon lower forward scatter (FSC) and side scatter (SSC) signals (i.e., smaller size and granularity) followed by gating off of CD3⁺ cells. Identification of positive cell populations was based upon using FMO controls.

To analyze the expression of exosomal surface markers, 4 μm aldehyde/sulfate latex beads (Thermo Fisher Scientific) were coated with anti-CD9 antibody (BD Biosciences, San Diego, CA; Cat: 555370) overnight and incubated with 30 μg of exosomes. The exosome-beads complexes were probed with human anti-CD81-PE (BD Biosciences; Cat: 555676) or human anti-CD63-PE (BD Biosciences; Cat: 557305) and data was acquired on a LSR II Flow cytometer (BD Biosciences). Data analysis was performed using the FloJo software (FlowJo version 10, Ashland, OR).