Human il 6 elisa ready set go

The concentrations of inflammatory and angiogenic makers in the serum and overnight effluent were determined using the ELISA technique. All samples were run simultaneously for each mediator to avoid intra- and inter-assay variations. Ang-1, Ang2, sTie-2 and VEGF levels were measured using commercially available Quantikine ® ELISA kits (R&D Systems Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions. IL-6 and IL-10 concentrations were determined by Human IL-6 ELISA Ready-SET-Go! (eBioscience) and Human IL-10 ELISA Ready-SET-Go! (eBioscience) respectively. Due to the concentrations of dialysate cytokines were influenced by ultrafiltration volume which was affected by peritoneal solute transport rate and dwell time, the dialysate appearance rate (AR) was calculated as dialysate concentration times the drained volume divided by the dwell time and expressed as picograms or nanograms per minute.

Plasma IL-6 levels were determined using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Human IL-6 ELISA Ready-SET-Go; eBioscience, San Diego, CA, USA) according to the manufacturer’s protocol. Samples were run in duplicate, and the mean was obtained.

IL-6 and IL-7 was determined in duplicate for diluted plasma samples using Human IL-6 ELISA Ready-SET-Go! (eBioscience) and Human IL-7 Quantikine HS ELISA kit (R&D Systems), respectively, according to manufacturer’s instructions. Samples were measured using the Infinite M200 ELISA reader (Tecan). Concentrations were calculated from the respective standard curves by applying 4-parametric logistic regression. Samples outside the detection range were set to the corresponding lower or upper range value.

The concentration of IL-6 in the drained PDF was determined with enzyme-linked immunosorbent assay (ELISA). All samples were run simultaneously and in duplicate to avoid intra- and inter- assay variations. The IL-6 concentration was measured by Human IL-6 ELISA Ready-SET-Go! (eBioscience^®, CA, USA). Due to the concentrations of dialysate cytokines were influenced by UF capacity which was affected by peritoneal solute transfer rate and dwell time, the dialysate appearance rate (AR) was calculated as dialysate concentration times the drained volume divided by the dwell time and expressed as pg (ng) per minute [pg (ng)/min].

For the quantitative assay of human VEGF-A, SKOV3ip1 cells (1 x 10⁵ cells) were cultured under serum-free medium in the presence or absence of IL-6 for 72 h. Anti-IL-6R antibody (10 μg/ml) or an equivalent amount of control IgG was concurrently applied. Thereafter, conditioned culture media were collected and stored at -80°C until analysis. Human VEGF-A Platinum ELISA kit (eBiosecience, SanDiego, CA) was used to determine the concentration of VEGF-A, in accordance with the manufacturer’s protocol, with a sensitivity of 7.9 pg/mL. For the quantitative assay of human IL-6 or human sIL-6R, 1 x 10⁵ of SKOV3ip1 cells and primary cells were cultured under serum-free medium for 72 h and these concentrations in culture medium was measured using Human IL-6 ELISA Ready-SET-GO with a sensitivity of 2 pg/mL or Human sIL-6R Instant ELISA (eBioscience) with a sensitivity of 10 pg/mL.

Plasma samples from HIV-infected and HIV^neg subjects were used to quantify human soluble CD14 (sCD14) using Quantikine ELISA Kit (R&D Systems, Cat # DC140), total IL-32 by DuoSet Human IL-32 (R&D Systems, Cat # DY3040), IL-32 alpha isoform using competitive Human Interleukin 32α (IL-32α) Elisa kit (MyBioSource, Cat # MBS750027), IL-6 using Human IL-6 ELISA Ready SET-Go (eBioscience, Cat # 88-706622), according to the supplier’s protocol. Cell-associated total IL-32 was measured by ELISA from cell lysates prepared as follows: total PBMCs from HIV⁺ or HIV^neg subjects were lysed by 1X RIPA buffer (Cell Signaling, Cat #9806) supplemented with protease inhibitors (Roche, Cat # 04 693 159 001 and 04 906 837 001) followed by quantification of total protein per lysate using the Bradford assay60 (link). ELISA measures from total cell lysate were normalized to the total protein in the lysate and plotted on graphs as IL-32 pg per 1μg of total cellular protein.