Dulbecco s pbs

Blood was drawn from mice via cardiac puncture with an EDTA-coated syringe. Thymus and spleens were excised and pushed through a 70-μm strainer, and bone marrow cells from both femurs and tibias were collected by centrifugation. Brains were harvested from mice perfused with 10 mL Dulbecco’s PBS (Gibco), minced into small pieces, and digested in RPMI 1640 (Gibco) with 300 μg/mL Liberase, and 100 U/mL DNase-I for 30 min at 37 °C. The tissue was then passed through a 70-μm strainer and mononuclear cells were enriched by 30% Percoll-gradient centrifugation. All samples were collected in Dulbecco’s PBS (Gibco) and were stored on ice during staining and analysis. Red blood cells were lysed in RBC Lysis Buffer according to the manufacturer’s protocol (BioLegend).

For the protein library generation approximately 30 million near confluent unstimulated adherent MM96L melanoma cells were trypsinised for 2 min and then harvested followed by three washes with Dulbecco’s PBS (Gibco, Waltham, MA, USA) with centrifugation at 800× g for 5 min between each wash. For SWATH quantification, 100,000 adherent mm96l melanoma cells were harvested in triplicate at different time points post-stimulation (unstimulated, 5 min, 30 min, 1, 6, 12, and 24 h) and were washed three times with Dulbecco’s PBS (Gibco, Waltham, MA, USA) with centrifugation at 800× g for 5 min between each wash. Stimulated and unstimulated MM96L cells were lysed on ice by resuspending the cells pellet in 100 mM TEAB (Thriethylammonium bicarbonate), 1% SDS (sodium dodecyl sulfate), 10 mM CHAPS, 5 mM MgCl₂ supplemented with 1× Roche Complete protease inhibitors. DNA and RNA were then degraded by the addition of 50 µL and 2 µL, respectively, of 50 mM Tris, 20 mM NaCl, and 2 mM MgCl₂ at 1 unit/µl of ultrapure Benzonase (Sigma, Munich, Germany) followed by incubation at 4 °C for 45 min with constant agitation. Protein concentration was determined using the Pierce BCA Assay (ThermoFisher Scientific, Waltham, MA, USA) following the manufacturer’s protocol.

Cell culture components obtained from Sigma (St. Louis, MO) include: bovine serum albumin (BSA, 30% solution), trypsin, penicillin streptomycin, MEM (phenol red free), sodium bicarbonate, fibronectin, fetal bovine serum (FBS), L-Glutamine. Dulbecco's PBS (1x without Ca2+ and Mg2+) was from Fisher Scientific (Houston, TX). BAECs were purchased from VEC Technologies (Rensselaer, NY) and grown in T-75 flasks with 10% FBS-MEM. Silicone sheets (Down Corning Corporation, MI) were used as the substrates for cell culture. BAECs were seeded onto the upper surface of silicone membranes of 0.020" thickness. First the membranes were laser cut to the geometrical specifications of the bioreactor, and then silicone substrates were washed for 20min with a gentle detergent and autoclaved for 30 minutes. A region delimited by an area of 1.5 x 4.5 cm on each substrate was coated using bovine plasma fibronectin (30μg/ml in MEM) for 1 hour at room temperature. The BAECs between passages 3 and 7 were plated at a density of 1.0x10⁵ cells/cm² onto the treated silicone substrates. ECs were grown using 10% FBS until confluency in a controlled environment (37C and 5% CO2 air). The EC monolayer reached confluency within four days.

Cell culture components obtained from Sigma (St. Louis, MO) include: bovine serum albumin (BSA, 30% solution), trypsin, penicillin streptomycin, MEM (phenol red free), sodium bicarbonate, fibronectin, fetal bovine serum (FBS), L-Glutamine. Dulbecco's PBS (1x without Ca2+ and Mg2+) from Fisher Scientific (Houston, TX).
BAECs were purchased from VEC Technologies (Rensselaer, NY) and grown in T-75 flasks with 10% FBS-MEM. Silicone sheets (Down Corning Corporation, MI) were used as the substrates for cell culture. BAECs were seeded onto the upper surface of silicone membranes of 0.020" thickness. First the membranes were laser cut to the geometrical specifications of the bioreactor, and then silicone substrates were washed for 20min with a gentle detergent and autoclaved for 30 minutes. A region delimited by an area of 1.5 x 4.5 cm on each substrate was coated using bovine plasma fibronectin (30μg/ml in MEM) for 1 hour at room temperature. The BAECs between passages 3 and 7 were plated at a density of 1.0x10⁵ cells/cm² onto the treated silicone substrates. ECs were grown using with 10% FBS until confluency in a controlled environment (37C and 5% CO₂ air). The EC monolayer reached confluence within four days.

Samples were lysed with RIPA buffer (Thermo Fisher, #89900). Protein concentration was determined by bicinchonic acid assay. 100 µg of protein from each sample was loaded onto a Criterion 10% Tris-HCl gel (Bio-Rad, Hercules, CA, cat #3451018) and electrophoresed. The proteins were then transferred to a PVDF membrane (Bio-Rad, cat #1620174) and blocked with 5% powdered milk (Bio-Rad, cat #1706404) in Dulbecco’s PBS (Thermo Fisher, cat #14190250) +0.1%Tween®20 (Sigma-Aldrich, St. Louis, MO, cat #P9416) for 1 h. The membrane was subsequently incubated with mouse-anti-human CD63 primary antibody (BD, cat #556019), at a 1:1000 dilution for 1 hour, and mouse-anti-human CD81 (Santa Cruz Biotechnology, cat #sc-166029), at a 1:1000 dilution for 1 hour. After washing the membrane, it was incubated with a goat-anti-mouse IgG-HRP secondary antibody (Santa Cruz Technology, cat #sc-2005) at a 1:10,000 dilution for 1 h. The membrane was then incubated with a 1:1 mixture of SuperSignal West Pico Stable Peroxide solution (Thermo Fisher, cat #34080) and Luminol Enhancer solution (Thermo Fisher, cat #34080) for 5 min. The membrane was visualized on Amersham Hyperfilm^TM ECL chemiluminescence film (GE Life Sciences, PA, cat #28906839).