Mouse igg1k

EVs were attached to 4 µm aldehyde/sulfate latex beads (Invitrogen) by mixing 30 µg EVs in 10 µL beads for 15 min at room temperature with continuous rotation. The suspension was diluted to 1 mL with PBS and incubated overnight with continuous rotation at 4 °C. The reaction was terminated with 100 mM glycine and maintained for 30 min at room temperature. Next, the solution was centrifuged for 3 min at 4000 rpm in a microcentrifuge and the resulting supernatant discarded. Beads were washed twice with PBS/0.5% BSA with centrifugation at 4000 rpm for 3 min. EV-bound beads were incubated with biotin-labeled anti-CD9 (catalog number: 13-0098, eBioscience, San Diego, CA, USA) for 30 min with continuous rotation at 4 °C and rewashed twice with PBS/0.5% BSA with centrifugation at 4000 rpm for 3 min. Next, beads were incubated with streptavidin conjugated phycoerythrin (SA-PE) (BioLegend) for 30 min with continuous rotation at 4 °C. Beads were washed three times with PBS/0.5% BSA and pelleted at 4000 rpm for 3 min, and the pellet was resuspended in 1 mL PBS/BSA. Antibody-stained EV-coated beads were analyzed on a flow cytometer (FACSCalibur, BD Biosciencs, Franklin Lakes, NJ, USA) and fluorescence compared with specific antibodies and relevant isotype controls (Mouse IgG1K, eBioscience).

HUVEC, HMEC-1 or HC-6014 were separated from their co-culture with Caki-1 cells. For separation by flow cytometry Caki-1 cells were marked with the CellTracker CM-Dil dye (Mo Bi Tec, Göttingen, Germany) prior to co-culture (detectable by FACScalibur, BD Biosciences, Heidelberg; FL2-H channel histogram analysis; 1 × 10⁴ cells/scan). Cells were detached, washed in FACS buffer (PBS, 0.5% BSA) and incubated for 60 min at 4°C with monoclonal antibodies (mAbs) directed against the following endothelial adhesion receptors: ICAM-1 (CD54, HA58), VCAM-1 (CD106, 51-10C9), E-selectin (CD62E, 68-5H11), P-selectin (CD62P, AK-4, all: APC labelled, anti-human, mouse IgG1 K, 20 μl, BD Pharmingen, Heidelberg, Germany), CD44 standard (CD44std, SFF-2, mouse IgG1 K, 1:20) and CD44 variants V3 (VFF-327v3, 1:10), V4 (VFF-11, 1:20), V5 (VFF-8) and V7 (VFFF-9, 1:20, all: mouse IgG1, 20 μl, eBioscience, Frankfurt, Germany). All CD44 antibodies were labelled with APC (Lightning-Link Allophycocyanin – XL Conjugation Kit, Innova Biosciences, Cambridge, UK). APC mouse IgG1, K (MOPC-21, 20 μl, BD Pharmingen, Heidelberg, Germany) served as isotype control. Receptor expression of HUVEC was measured by flow cytometry using FACScalibur (BD Biosciences, Heidelberg; FL4-H channel histogram analysis; 1 × 10⁴ cells/scan). Data were expressed as mean relative fluorescence intensity (MFI).

PBMCs were pretreated with different concentrations (1.25, 2.5 and 5 μg/mL) of anti-MCP-1 antibody (clone 5D3-F7; eBioscience) or an isotype control (mouse IgG1 K; eBioscience). The following day (20 hpi) the cells were infected with CHIKV at MOI 1, 5 and 10. 2 hpi the viral inoculum was removed and replaced by fresh medium containing the corresponding antibody. At 24 hpi, cell-free supernatant was collected and viral production was determined by plaque assay and qPCR.