Pe mouse igg1 p3

Eighty microliters of blood were collected by tail vein incision in a tube containing 20 μl of 3.8% sodium citrate. Erythrocytes were lysed by adding 600 μl of erythrocyte lysis solution (Qiagen, Valencia, CA) for 5 min in agitation. Then blood was centrifuged at 260 g for 5 min and the pellet was resuspended in 10 ml of cold PBS. Cells were centrifuged at 260 g for 5 min and then resuspended in 100 μl of FACS buffer (1% FBS in PBS) containing the appropriate antibodies. Cells were stained at 4°C in the dark for 1 h and then pelleted after adding 1 ml of FACS buffer. Cells were resuspended in 500 μl of FACS buffer and analyzed at BD FACSCalibur flow cytometer. B-lymphocytes were stained with APC Cd45 (30-F11, #103112, 1:100; BioLegend), FITC Cd19 (6D5, #115506, 1:100; BioLegend), and PE Cd39 (5F2, #12-3390-80, 1:100; eBioscience). APC rat IgG2b (RTK4530, #400611, 1:100; BioLegend), FITC rat IgG2a (R35-95, #554688, 1:100; BD Biosciences), and PE mouse IgG1 (P3.6.2.8.1, #12-4714-41, 1:100; eBioscience) were used as isotype controls. Thirty thousand events from lymphocyte gate were acquired in each analysis.

VSMCs were isolated from murine aorta as previously described (27 (link)). In order to isolate enough cells, three to four aortas were pooled together. Briefly, aortas were dissected and the adventitial tissue and fat were removed. Then aortas were cut into 1–2 mm rings and put in digestion with Collagenase type II (Gibco) for 6 h (7.5 mg of Collagenase II in 5.5 ml of culture medium: DMEM containing 10% FBS, 1× penicillin/streptomycin, and 1% glutamine). The lysate was pelleted and the cells were resuspended in 700 μl of culture medium and cultured at 37°C, 5% CO₂. After 5 days, the medium was replaced and, once confluent, cells were trypsinized and expanded. Cells were cultured up to the third passage. The purity of VSMCs was checked by immunofluorescence staining with anti-actin, α-Smooth Muscle-Cy3 (1A4, #C6198, 1:5,000; Sigma-Aldrich). Cd39 expression was evaluated by FACS analyzing 30,000 cells using PE Cd39 (5F2, #12-3390-80, 1:100; eBioscience) and PE mouse IgG1 (P3.6.2.8.1, #12-4714-41, 1:100; eBioscience) as isotype control.