Imagestream x mark 2

The isolated EV fraction was analyzed using an Image Stream X Mark II imaging flow cytometer (EMD, Millipore Sigma, Seattle, WA). Protein surface markers for different EV subtypes were utilized to confirm presence and estimate subpopulation proportions of the circulating EVs. Proteins chosen for analysis represent markers of exosomes (CD9), microvesicles (vesicle‐associated membrane‐3 (VAMP3)), and apoptotic bodies (thrombospondin‐1 [THSD1]; Akers et al., 2013 (link); Conkright, Beckner, Sahu, et al., 2022 (link)). α‐Sarcoglycan (SGCA) has also been implicated as a marker of muscle‐derived EVs. 250 μL of isolated EVs were added to 50 μL blocking buffer (3% BSA in sterile‐filtered PBS) and placed on a rocker to block for 1 h. at RT. Samples were then stained with the following antibodies and dilutions: anti‐human CD9 Alexa Fluor 700 (1:300 dilution; Novus Biologicals, CO), anti‐human VAMP3 Alexa Fluor 405 (1:300; Novus Biologicals), anti‐human thrombospondin (THSD‐1) Alexa Fluor 594 (1:100; Novus Biologicals), alpha sarcoglycan (SGCA) FITC (1:400; Biorbyt, St Louis, MO). Samples were incubated overnight at 4°C and run through the flow cytometer the following day. Several control conditions were utilized to establish proper gating, to correct for fluorescence carryover, and to ensure quality data was obtained. Briefly, buffer only and antibody only controls were run and single‐stained compensation controls (UltraComp eBeads Plus; Invitrogen, Waltham, MA) were used to correct for fluorescence carryover between channels. Fluorescence minus one (FMO) controls were run to establish final gating strategies for the fluorescent channels. All controls were prepared according to the staining protocol above, except for the compensation controls, which were prepared according to the manufacturer's instructions.
ImageStream settings were consistent for all samples: 60× magnification, high gain mode, fluidics set to high sensitivity and slow speed, auto‐focus, and auto‐centering. Laser voltages were adjusted to keep Raw Max pixels below 4e^3 and were as follows: 405 nm 150 mW, 488 nm 125 mW, 561 nm 150 mW, 642 nm 150 mW, and SSC 70 mW. All timepoints for a single subject were prepared and analyzed on the cytometer on the same day and each sample was run for 3 min.
Flow cytometry data were analyzed using the IDEAS 6.2 software (MilliporeSigma, Burlington, MA) as previously described (Conkright, Beckner, Sterczala, et al., 2022 (link)) with slight modifications. ImageStream SpeedBeads were gated out of all samples using a histogram of the side scatter channel and only gating events less than the high side scatter peaks indicative of single and clumped SpeedBeads. Quantification of scatter and fluorescent intensities was established using a masking strategy described by Tertel et al. (2020 (link)) optimized for EV analyses. Positive events in each EV subpopulation were then gated according to their appropriate fluorescent channel and the gates were adjusted using each FMO controls when appropriate. Template files were utilized so that all samples run on the same day were gated in the same manner. Batch analysis was done using the appropriate templates and a final statistical report was generated for each sample containing percentage gated of the EV events gate.

For Imagestream analysis, live TILs at 1 × 10⁷ cells per ml were run at 100 cells per second on the ImageStreamX MarkII (Merck Millipore). TILs were stained with relevant antibodies for CD4, CD8, IL-2Rα, IL-2Rβ, IL-2Rγ_c and DAPI. Single stained cells were used as compensation controls. Images were captured at 60× magnification. Data were analysed using the ImageStream Data Analysis and Exploration Software (IDEAS). Colocalization was calculated based on Bright Detail Similarity score, a log-transformed Pearson’s correlation coefficient computed by Amnis.