Mouse tnf elisa set

Tyrode-HEPES buffer was purchased from BioSolution (Suwon, Republic of Korea). Phosphate buffered saline (1×) without calcium and magnesium (#17-516F) was purchased from Lonza (Basel, Switzerland). Acid-citrate-dextrose (ACD) solution (#C3821), prostaglandin E1 (PGE1; #P5515), neuraminidase (sialidase) from Clostridium perfringens (#11585886001), and OS (#SML1606) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Erythrina crista-galli lectin (#FL-1141-5), SNA (#FL-1301-2), and RCA-I (#FL-1081-5) were purchased from Vector Laboratories (Burlingame, CA, USA). Maackia amurensis lectin II (#21511103-1) antibody was purchased from bioWORLD (Dublin, OH, USA). Anti-CD41 (#11-0411-82) and anti-CD62P (#17-0626-82) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-fibrinogen antibody (ab34269) was purchased from Abcam (Cambridge, UK). Monoclonal antibodies against JAK2 (#3230), phospho-JAK2 (#3776), STAT3 (#12640), and phospho-STAT3 (#9145) were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse Thrombopoietin Quantikine ELISA Kit (#MTP00) was purchased from R&D Systems (Minneapolis, MN, USA). Mouse TNF ELISA Set (#555268) and Mouse IL-6 ELISA Set (#555240) were purchased from BD Biosciences (San Jose, CA, USA).

Culture media collected from RAW 264.7 mouse macrophages 6 h post-treatment of either Naïve EVs, LPS (100ng/mL), LPS+eIL-10(500ng) were tested for mouse IL-6, TNFα and IL-1β using the Mouse IL-6 Quantikine ELISA (M6000B, Minneapolis, MN, USA) Mouse TNF ELISA Set (555268, BD Biosciences, San Jose, CA) and Mouse IL-1β ELISA Set (561667, BD Biosciences, San Jose, CA) respectively. Standard curves were developed using duplicate samples of known-quantity r proteins that were provided by the manufacturer. Sample concentrations were determined by relating the absorbance of the samples to the standard curve using linear regression analysis.

Macrophages from control or drug-treated mice were standardly cultured at 37 °C and 5% CO₂, at a concentration of 2 × 10⁶ cells per well in 2 mL of RPMI1640 with 5% fetal bovine serum (FBS, Biowest, Nuaillé, France). Macrophages in some wells were stimulated with lipopolysaccharide (LPS, 200 ng per well, BIO-Whittaker, Walkersville, MD, USA). The resulting supernatants were collected for evaluation of IL-6, and tumor necrosis factor-alpha (TNFα) concentrations after 24 h, and for IL-10 and transforming growth factor-beta 1 (TGF-β1) measurement after 48 h of the culture. Cytokine concentrations were measured in supernatants stored at −80 °C, in enzyme-linked immunosorbent assay (ELISA), according to manufacturer procedures. The following OptEIA™ kits were used: mouse TNF ELISA Set (catalog No 558534), mouse IL-6 ELISA Set (catalog No 555240), mouse IL-10 ELISA Set (catalog No 555252), all from (BD Biosciences, San Diego, CA, USA). In addition, TGF-β1 concentration was measured with the use of mouse TGF-beta1 Platinum ELISA Test (catalog No BMS608/4) from (eBioscience Inc., San Diego, CA, USA).