Txb2 elisa kit

The level of TXB₂ in the culture medium was measured by TXB₂ ELISA kit (Cayman Chemical Company, Ann Arbor, MI, USA), whereas TGFβ3 in FF was determined by TGFβ3 ELISA kit (R&D Systems, McKinley Place, MN, USA). For determining TXB₂ in the culture medium, the GCs (1 × 10⁵/well) were seeded in a 48-well culture plate for 24 h. After brief starvation overnight, cells were treated with the indicated concentrations of TGFβ3 for 24 h. At the end of incubation, the culture medium was collected and centrifuged, and the TXB₂ levels were measured according to the manufacturer’s protocol. The enzymatic reaction produced a yellowish color that strongly absorbed light at 412 (TXB₂) or 450 (TGFβ3) nm. For measuring TXB₂ level, the intensity of this color was directly proportional to the amount of TXB₂ tracer bound to the well and inversely proportional to the amount of free TXB₂ present in the well during the incubation. The concentration of TXB₂ in the GC culture medium and TGFβ3 in the FF were calculated from their respective standard curve.

Patients with type 2 diabetes mellitus (T2DM) were recruited, from 2011 to 2014, at the Metabolic Disease Hospital of Tianjin Medical University, Tianjin Medical University General Hospital, the Tianjin People's Hospital, and the Eye Hospital of Tianjin Medical University (Wei et al, 2016 (link)), and their retinal complications were independently diagnosed by two experienced ophthalmologists. T2DM was defined as fasting plasma glucose ≥ 7.0 mmol/L, and/or 2‐h oral glucose tolerance tests (OGTT) ≥ 11.1 mmol/L, or the use of anti‐diabetic medicine. Serum samples were collected for medical tests and written consent was obtained from all patients. The procedure was approved by the Human Ethics Committee of Tianjin Medical University (TMUhMEC2020034) in accordance with the Helsinki declaration and the principles set out in the Department of Health and Human Services Belmont Report. Serum levels of 15‐keto‐dihydro‐PGF_2α and TXB₂ were determined using the 13, 14‐dihydro‐15‐keto PGF_2α ELISA Kit (Cayman, 516671) and TXB₂ ELISA Kit (Cayman, 501020), respectively, according to the manufacturer's instructions.

Leukotriene B₄ (LTB₄), Prostaglandin E₂ (PGE₂) and Thromboxane B₂ (TXB₂) were quantified by the LTB₄ ELISA, PGE₂ Monoclonal ELISA and TXB₂ ELISA kits, respectively, all from Cayman Chemical (Ann Arbor, MI, USA).

Human chemokines were detected by using a BD™ CBA Human Chemokine kit (BD Biosciences). The BD™ CBA Mouse Inflammation kit (BD Biosciences) was used to detect systemic chemokines and interleukins in the mouse plasma. Keratinocyte Chemokine (KC)/C-X-C motif chemokine ligand 1(CXCL1) was detected by using the LEGENDplex™ Mouse Anti-Virus response panel (Biolegend). Mouse myeloperoxidase (MPO) was quantified by using a MPO Mouse ELISA kit (HycultBiotech). leukotriene B4 (LTB₄), prostaglandin E2 (PGE₂), and thromboxane B2 (TXB₂) were quantified by the LTB4 Enzyme-Linked Immunosorbent Assay (ELISA), PGE2 Monoclonal, and TXB2 ELISA kits, all from Cayman Chemical. All assays were performed according to the manufacturer’s recommendations.