Cd144 apc

Flow cytometry was used to determine MP origin using a custom-built BD FACSAria II (BD Biosciences, CA). Forward scatter area (FSC-A) and side scatter area (SSC-A) were set to log scale and MPs were gated based on their FSC-A/SSC-A profile and in relation to platelets in fresh plasma. MP pellets were resuspended in 1× 0.22 μm-filtered annexin V binding buffer (BD Biosciences) and 100 μl of this was used for staining. MPs were stained in the dark (15 min, room temperature) with annexin V-FITC (1.57 μg/ml), CD41-PE-Cy5 (0.12 μg/ml), CD11b-PE-Cy7 (7.9 μg/ml), CD144-APC (4.1 μg/ml), and CD235a-PB (7.7 μg/ml) as markers of MPs, platelets, monocytes, endothelial cells, and erythrocytes, respectively (all antibodies from BioLegend, CA). Data were exported from the FACSDiva™ software (version 6) and subsequently analyzed in FlowJo software (version 9.6.4; Tree Star Inc., OR).

Fresh mouse kidney cortexes were digested in 2 mL of protease solution — 5 mM CaCl₂, 10 mg/mL Bacillus licheniformis protease (Creative Enzymes, NATE0633), and 125 U/mL DNase I (Roche) in Dulbecco’s PBS — on ice for 30 minutes. The lysates were filtered through 70 μm cell strainers. Single-cell suspension was collected by rinsing with FACS buffer. Suspensions were lysed with red blood cell lysis buffer (Quality Biological) to remove red blood cells. After washing with 1× PBS, 1 × 10⁶ cells were resuspended in 100 μL of FACS buffer and preincubated with anti–mouse CD16/32 antibody (BioLegend, 101302) for blocking prior to labeling with CD144-APC (BioLegend, 138012) and CD184-BV421 (BD Biosciences, 562738) antibodies for 30 minutes at room temperature. Cells were then washed with FACS buffer and analyzed on BD LSR II Violet at the University of Iowa Flow Cytometry Facility. All flow cytometry results were analyzed using FlowJo software (Version 9).