Chapter 4 102 dark overnight (dilution by 0.2-μm-pore-filtered Dulbecco’s phosphate-buffered saline; fDPBS). IgG1-Alexa488 and IgG1-APC were used as isotypes. The stained sample was treated with 6 µL of 10% (v/v) Triton-X-100 at room temperature for 30 min to lyse biological structures, such as EVs. Acquisition settings of ImageStreamx MkII Multispectral IFCM was performed using Amins ImageStream MKII (ISx, Luminex, Seattle, WA, USA), with data acquisition by software INSPIRE® (version: 200.1.681, EMD Millipore). INSPIRE® runs a quality control procedure with built-in ASSIST® as the daily startup procedure. The settings of INSPIRE® in the acquisition: low-speed (velocity: 40 mm/s) & high sensitivity, 6 µm core diameter, 60X magnification, 488-nm laser power at channel 02 (Ch02) detecting Alexa488: 200 mW; 642-nm laser power at channel 05 (Ch05) detecting APC: 150 mW, 1.25 mW laser power at channel 06 (Ch06) detecting side scatter (SSC), and activated channel 04 (Ch04) for detection of bright field. To monitor sample flow and maintain the camera’s focus in ISx, polystyrene speed beads (catalog no. 400041, Luminex) were loaded together with samples at 15%. Each acquisition lasted 180 s, the“Prim” function was clicked once for loading samples fast, and“Remove Speed Bead” was unchecked to maintain speed beads during acquisition. IFCM analysis Raw image files acquired from INSPIRE® were analyzed by ISx Data Exploration and Analysis Software (IDEAS® 6.2, EMD Millipore). “Mask” define a specific area within images to select and quantify pixels. Masks in Ch01 to Ch06 pictures were labeled from M01 to M06, and Mask Combined (MC) includes M01 to M06. Different functions can be applied to each Mask to adjust the position and region of selected pixels. Mask with the“Intensit” function contains pixels showing higher intensity than the backgrounds. The Mask with the“Pea” function (model: Bright) identifies the brightest pixels with the peak intensity in each event, designed for fluorescent spot recognition.28 The intensity threshold of the Peak Mask is determined by its cell-to-background ratio. The name of M02 in Ch02, with Peak function, Bright model, and cell-to-background ratio 1, is simplified as Peak (M02, Ch02, Bright, 1). Masks, Peak (M02, Ch02, Bright, 1), and Peak (M05, Ch05, Bright, 1) were used to include the pixels of Alexa488+ and APC+ fluorescent spots. By using the Boolean logic function, Peak masks applied for Ch02 and Ch05 were combined as“Peak
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