Dynal mpc s

2-(N-morpholino)ethanesulfonic acid (MES) was purchased from EMD-Calbiochem (Gibbstown, NJ). 1-Ethyl-3-(3-methylaminopropyl)carbodiimide (EDC), gluteraldehyde, hydroxylamine hydrochloride, Dithiothreitol (DTT) potassium phosphate dibasic, pyridine (99.8%), sodium azide, sodium cyanoborohydride, sodium chloride, tryptone, yeast extract, glucose, β-mercaptoethanol, EDTA, Isopropyl β-d-1-thiogalactopyranoside (IPTG), trizma base, calcium chloride, glycerol and sodium phosphate monobasic, quercetin, vitexin, naringenin, orientin were obtained from Sigma–Aldrich Chemical Co. (Milwaukee, WI). Species verification of lot number 401255 (T. foenum-graecum) was from HerbPharm (Williams, OR). N-Hydroxysulfosuccinimide (Sulfo-NHS) was from Pierce (Rockford, IL). Solutions were prepared using purified water from a Millipore MilliQ system (Millipore Corporation, Bedford, MA). BcMag amine-terminated magnetic beads (50 mg/mL, 1 μm) were purchased from Bioclone, Inc. (San Diego, CA). The manual magnetic separator Dynal MPC-S was from Invitrogen Corporation (Carlsbad, CA).

rhBri2 BRICHOS domain was immobilized via a previously published method, with slight modifications [17 (link)–19 (link)]. Briefly, 25 mg of BcMag magnetic beads (MB) were placed in a 2 mL microcentrifuge tube. The MBs were washed three times with 1 mL of coupling buffer (pyridine [10 mM, pH 6.0]) and were suspended in 1 mL of 5 % glutaraldehyde solution in coupling buffer and rotated for 3 h at room temperature (RT). After separation using the manual magnetic separator (Dynal MPC-S, Invitrogen Corporation, Carlsbad, CA), the MBs were washed three times with 1 mL of coupling buffer. A suspension of 200 μg of (rh) Bri2 BRICHOS (90–236) domain in 500 μL of coupling buffer containing 1 % sodium cyanoborohydride was added to the activated MBs. The reaction was left for 7 days at 4 °C with gentle rotation. The supernatant was discarded and 200 μL of coupling buffer containing 100 mM hydroxylamine and 2.5 % cyanoborohydride was added. The mixture was shaken overnight at RT with gentle rotation. The supernatant was discarded and MBs were washed 3x with 1 mL of 1xPBS (containing 0.02 % NaN₃). The control hydroxylamine-coated (NT)-MB (Control-MB) were made in the same manner but without the addition of rhBri2.

Retinas were dissected, minced, and incubated in Dulbecco modified Eagle medium (4.5 g/L glucose with L-glutamine) containing 200 U/mL collagenase IV (Invitrogen) and 2.4 U/mL Dispase (17105–041; Life technologies) for 30 minutes at 37°C on agitation. To obtain a single-cell suspension, cells were first passed through a 25-G needle, then filtered through a cell strainer (70-μm pore size; BD Falcon) centrifuged at 200g for 5 minutes at 4°C. Cells were then resuspended in cold washing buffer (0.1% BSA, 2 mmol/L EDTA pH 7.4 in PBS), and incubated with biotinylated iB4 (1:12.5; Vector Laboratories) for 30 minutes on ice with agitation and after that incubated with streptavidin-coupled magnetic beads (Dynabeads MyOne Streptavidin T1, 656.02; Dynal Biotech ASA) for 30 minutes on ice with gentle agitation. The beads were separated from the solution using a magnetic particle concentrator (Dynal MPC-S; Invitrogen), and the cells in the supernatant were kept as the non-EC fraction. Non-EC fraction and washed beads were pelleted at 200g for 5 minutes, and the supernatant was removed. The purified ECs and non-ECs were snap-frozen in liquid nitrogen and stored at −80°C until use.

We harvested mouse lungs at P21, minced them and incubated them in 5 mL Dulbecco's modified Eagle's medium containing 2 mg/mL collagenase I (Invitrogen) for 45 min at 37 °C with shaking every 15 min followed by filtering through a 40-μm nylon mesh (BD Falcon). The cells were then centrifuged at 1,000g for 5 min at 4 °C, resuspended in buffer 1 (0.1% bovine serum albumin, 2 mM EDTA, pH 7.4, in PBS), and incubated with anti-rat immunoglobulin G–coated magnetic beads (Invitrogen) precoupled with rat anti-mouse platelet/endothelial cell adhesion molecule-1 (PECAM-1; MEC13.3, BD Pharmingen, 553370) for 30 min at 4 °C in an overhead shaker. Beads were separated from the solution with a magnetic particle concentrator (Dynal MPC-S, Invitrogen). The beads were washed five times with buffer 1 and centrifuged for 5 min at 1,000g, and the supernatant was removed. The purified endothelial cells were then cultured in ECGM-2 (Promocell). For western blot analysis, lung endothelial cells (2 × 10⁵) were seeded in 60-mm dishes and cultured for 24 h in ECGM-2 at 37 °C and 5% CO₂.