Fc receptor blocking agent

Cells were trypsinized, resuspended in PBS supplemented with 2% BSA, incubated with Fc receptor blocking agent (Biolegend, 101302), and stained with fluorescently labeled antibodies for 30 min on ice: FITC-conjugated CD8α, CD4, and PE-conjugated NK1.1, CD45, and PerCP/Cyanine5.5-conjugated CD3, Gr-1, and BV421-conjugated F4/80 and BV510-conjugated CD11b, and APC-conjugated CD107a, CD54, and PE/CY7-conjugated PD-1, PD-L1. For intracellular detection, cells were stimulated with PMA and ionomycin (BD Biosciences) for 6 h, fixed and permeabilized with Cytofix/Cytoperm Kit (BD Biosciences) and stained with specific antibodies: BV510-conjugated IFNγ. For apoptosis assay, an annexinV/PI apoptosis detection kit was used according to the manufacturer’s protocol (Biovision). The data were analyzed with FlowJo software (version 10.5; Tree Star).

MSCs were detected the expression of specific surface markers, including PE anti-mouse Sca-1, PE anti-mouse CD90.2, PE anti-mouse CD29, PE anti-mouse CD44, PE anti-mouse CD45, APC anti-mouse CD31, APC anti-mouse CD34, and APC anti-mouse CD117 (Biolegend, San Diego, CA).
After MSCs were treated with 3 mM NMDA (Sigma-Aldrich) or 50 μM MK801 (Sigma-Aldrich) for 24 h, APC anti-mouse C-X-C chemokine receptor type 4 (CXCR4; Biolegend) was used for surface staining to study the effect of NMDA receptor activation on CXCR4 expression in MSCs.
MSCs were harvested and then blocked with Fc receptor blocking agent (BioLegend) for 10 min on ice. The antibodies were added and incubated for 30 min at 4°C in dark. The cells were then washed with 0.1% BSA and fixed with 1% paraformaldehyde in PBS. Cell surface staining was analyzed using a FACSCanto II (Becton Dickinson, Franklin Lakes, NJ) within 24 h of staining. FlowJo software version 7.6.1 (FlowJo, Ashland, OR) was used for data analyses.

Aortic tissues were minced and digested in digestion solution containing Elastase (Worthington) (0.25 mg ml⁻¹) and LiberaseTH (Roche) (0.025 mg ml⁻¹). Digested tissues were further dissociated with a 21-G needle. Remaining deposited debris was removed and the supernatant was collected after filtering through a 40-μm cell strainer. Cells were suspended in PBS containing 3% FBS, and nonspecific binding of the antibodies to Fc receptors was blocked using an Fc receptor-blocking agent (1:50) (BioLegend). Cells were stained with APC-anti-mouse CD11b (1:150), PE anti-mouse F4/80 (1:20), APC-Cy7 anti-mouse Ly6c (1:300) and Alexa488 anti-mouse CD206 (1:50) (BioLegend). The LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen) was used to label dead cells. After washing, cells were analysed using BD FACSVerse. Cell sorting was performed by BD FACSAria II. The data were analysed by Flo Jo software (Tree Star).