Anti cd31 fitc antibody

To determine effects of chemotherapy and serological conditioning on HCAEC apoptosis, a panel of monoclonal antibodies (mAbs) were used. HCAECs were incubated for 30 min at 4°C with anti-CD31-FITC antibody (BioLegend, United States) and anti-Annexin V-PerCP-Cy5.5 antibody (BD Biosciences, United States). For subsequent intracellular staining, endothelial cells were incubated for 20 min at 4°C with 100 µL BD fixation and permeabilization solution (BD Biosciences, United States). HCAECs were then incubated for 30 min at 4°C with anti-cleaved caspase-3-V450 antibody (BD Biosciences, United States). Lastly, HCAECs were washed twice with 100 µL perm wash buffer (BD Biosciences, United States) and resuspended in 200 µL PBS (Invitrogen, Thermofisher, United States).
After performing calibration and fluorescence compensation with CS&T beads (BD Biosciences, United States) and compensation beads (BD CompBeads Set, BD Biosciences, United States), respectively, samples were analysed using the High Throughput System on a 12-colour flow cytometer (FACS Celesta, BD Biosciences, United States). Data were acquired using FACSDiva 6.0 Software (BD Bioscience, United States), with the sample acquisition stopping gate set to 10,000 events.

Human lung microvascular endothelial cells (HLMVECs), THP-1 cells, MLE12 cells, and Raw 264.7 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). HLMVECs were cultured with EGM-2MV microvascular endothelial growth medium containing 5% fetal bovine serum (FBS) at 37 °C in 5% CO₂. LPS (E. coli O55:B5), thrombin, and the anti-β-actin antibody were purchased from Sigma–Aldrich (St. Louis, MO, USA). Antibodies against VE-cadherin, USP40, ICAM1, VCAM1, Lamin A/C, and GAPDH and immobilized protein A/G beads were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against pMLC, MLC, the Flag tag, the V5 tag, pLimk1, Limk1, p-cofilin 1, cofilin 1, pIκBα, p65, p-p65, HSP90β, K63-linked ubiquitin, ubiquitin, and acetylated lysine (AcK) were purchased from Cell Signaling (Beverly, MA, USA). ELISA kits for quantifying TNFα, IL-6, and IL-8 were obtained from eBioscience (San Diego, CA), and mouse IL-1β, KC/CXCL1 ELISA kits, and human recombinant TNFα protein were purchased from R&D Systems (Minneapolis, MN, USA). The anti-CD31-FITC antibody was obtained from Biolegend (San Diego, CA, USA). GeneJet™ reagent and GeneMute siRNA transfection reagent were purchased from SignaGen (Frederick, MD, USA). All materials used in the experiments were of the highest grade commercially available.