Human esm1 elisa kit

The blood samples were taken in 7.00-9.00 a.m. after participants fasted at least 8 hours. In order to obtain sera, samples were left to clot within 30 minutes and then centrifuged at room temperature for 10 minutes at 3000xg.
Serum endocan levels were determined by using an enzyme-linked immunosorbent commercial assay (ab213776 – Human ESM1 ELISA Kit, Abcam, Cambridge, UK).
Immunoturbidimetric method was used for determination of HbA1c in samples of a whole blood [Roche Cobas c501 chemistry analyzer (Roche Diagnostics GmbH, Mannheim, Germany)]. Serum glucose and lipid parameters (i.e., TG and HDL-c) were measured spectrophotometrically, on the same autoanalyzer as HbA1c. Serum hsCRP level was obtained by nephelometric assay (Behring Nephelometer Analyzer, Marburg, Germany).

PD sheets and PD sheets-mini fabricated as described above were used. PD sheets collected by hooking on paper rings were cultured for 24 h in 3 mL of DMEM/F12 supplemented with 5% FBS and 1% AB in an adherent culture dish. PD sheets-mini were harvested from RepCell™ plates after the plate was left at room temperature for more than 30 min. PD sheets-mini were suspended in 3 mL of DMEM/F12 supplemented with 5% FBS and 1% AB, and cultured for 24 h in a culture dish. Each supernatant was collected and centrifuged at 15,000× g for 10 min to remove cell debris. The concentrations of TGF-β1, MIA, ESM-1, DKK-1, MCP-1, MMP-3, and MMP-13 were measured using the enzyme-linked immunosorbent assays (ELISAs) described below.
TGF-β1, MIA, and ESM-1 concentrations were measured using their respective single-sample ELISA kits: Human TGF-β1 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA), MIA ELISA (Roche), and Human ESM-1 ELISA Kit (Abcam, Cambridge, UK). The other humoral factors were measured using Quantibody Multiplex ELISA Array (RayBiotech Life, Inc., Norcross, GA, USA).
The signal detected for blank medium containing 5% FBS was subtracted to adjust for proteins contained in FBS. Measurements were repeated at least twice for each donor, and averages were used. The amount of humoral factor per area of the culture surface was calculated.

EA.hy926 cells and HUVECs were seeded in 24-well plates at 50,000 and 25,000 cells per well, respectively. ECs were then incubated for 24 h at 37°C and 5% CO₂ prior to incubation in 1 ml of control medium or macrophage-conditioned media, as indicated in Section 2.3. In parallel, 1 ml of each corresponding conditioned medium was incubated at 37°C during the same period (24 h). Supernatants were harvested and centrifuged at 200g for 5 min at 4°C. Supernatants were stored at −70°C and used afterward for cytokine quantification. In parallel, ECs were lysed with 200 µl of NaOH 0.5 N and frozen at −20°C. The concentration of IL-6 and IL-8 in supernatants was analyzed using human quantikine ELISA kits (R&D Systems), which were used according to the manufacturer’s indications. The concentration of endothelial cell–specific molecule 1 (ESM1) protein in supernatant was analyzed using a human ESM1 ELISA kit (ab213776, Abcam) and used according to the manufacturer’s indications. To quantify only the protein secreted by ECs, the proteins contained in the macrophage supernatant were subtracted from each corresponding EC supernatant. Cytokine concentrations (picograms per milliliter) were normalized by total protein concentration (micrograms) contained in ECs and macrophages determined by the Folin method after 200 µl (ECs) or 1 ml (macrophages) of NaOH 0.5 N lysis.