Chapter 6 166 Class I molecules compared to CD63 (~3-fold difference) (Supplementary Figure S1 online). mAbs used in this study were CD9–APC, clone HI9a (6 µg/mL, Biolegend, San Diego, USA), and HLA-A3-BV421, clone GAP-A3 (200 µg/mL, BD Biosciences, New York, USA). Matched isotype controls were IgG1, k-APC, clone MOPC-21 (200 µg/ mL, BioLegend), and IgG2a, k-BV421, clone G155-178 (200 µg/mL, BD Biosciences). Specificity of the anti-HLA-A3 mAbs was confirmed by Luminex single antigen assay (Supplementary Data S1 online). Prior to staining, mAbs and isotypes were centrifuged at 16,000 x g for 10 minutes at room temperature to remove potential mAb clumps (to reduce false-positive signals from analysis), and were diluted in 0.20 µm filtered PBS (fPBS) before staining (final concentrations: 200 ng/mL). Staining was performed by addition of the diluted mAbs/isotypes to 30 µL of PPP followed by a pre-determined volume of fPBS (Vtot = fPBS + sample + mAbs = 130 µL) followed by O/N incubation at 4 0C. All samples were brought to a total volume of 380 µL using fPBS before IFCM measurements. Controls To ascertain EV measurements the following controls were applied, as recommended by the MIFlowCyt-EV framework 22: buffer only, buffer with reagents, unstained samples, isotype controls, and detergent treatment, which aims to disrupt the membranous structure of EVs thereby allowing discrimination between membrane-enclosed vesicles (which lyse upon detergent treatment) and other protein complexes (which are unaffected by detergent treatment). Detergent treatment was performed by adding 20 µL of 10% (V/V) TritonX-100 to the samples followed by 30 minutes of incubation at room temperature prior to acquisition. Data acquisition All samples were acquired on an ImageStreamX MkII instrument (ISx; Luminex). Settings as extensively described elsewhere were used 23. In brief, lasers were turned on as applicable per fluorophore and set to their maximum power (405 nm : 200 mW, 642 nm :150 mW) with the exception of the 785 nm SSC laser (1.25 mW). High Gain mode – an upgrade of the IFCM that increases the photonic sensitivity and object detection of the system – was activated. Data was acquired over fixed time periods – to standardize among samples – of 180 seconds using the 60x objective with fluidics set to ‘low speed / high sensitivity’. This resulted in a flow
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