Mouse tgf beta 1 picokine elisa kit

MEF cells (2.5 × 10⁵) were cultured at confluence in 350 µl serum‐free DMEM or DMEM containing 10% FBS for 24 hr. TGF‐β1 levels in the medium were determined using Mouse TGF Beta 1 PicoKine™ ELISA Kit (BosterBio) according to the manufacturer’s protocol. Briefly, 100 µl cell culture supernatant was activated with 20 µl of 1 M HCl for 10 min at room temperature, followed by pH neutralization with 1.2 M NaOH–0.5 M HEPES. The activated supernatant (100 µl) was added to 96‐well plate precoated with anti‐TGF‐β1 antibody and incubated at 37°C for 90 min. The supernatant was discarded and 100 µl of biotinylated anti‐TGF‐β1 antibody was added to each sample for 60 min incubation at 37°C. Contents in the plate were discarded and the plate was washed three times with PBS, followed by incubation with 100 µl ABC working solution for 30 min at 37°C. After five times wash with PBS, 90 µl TMB color‐developing agent was added. After 30 min of incubation at 37°C, the reaction was terminated by the addition of 100 µl stop solution. Absorbance at 450 nm was recorded using Synergy H1 microplate reader (BioTek, Dubai, U.A.E). TGF‐β1 concentration was extracted based on the standard curve of recombinant TGF‐β1.

Antibodies of myosin light chain 9 (Myl9; ab64161), Itga11 (ab198826) were from Abcam (Cambridge, UK). Antibodies against β‐actin (clone AC‐74), diphenyleneiodonium chloride (DPI; D2926), SB431542 hydrate (S4317), N‐acetyl‐l‐cysteine (A7250), Dulbecco’s modified Eagle’s medium (DMEM) high glucose (D5671), fetal bovine serum (FBS; F0804), and penicillin–streptomycin (P0781) were from Sigma‐Aldrich (Taufkirchen, Germany). Antibodies of mouse immunoglobulin G (IgG) horseradish peroxidase (HRP; 62‐6520), rabbit IgG HRP (65‐6120), smooth muscle actin antibody (PA5‐19465), Maxima SYBR Green qPCR Master Mix (K0252), RevertAid First Strand cDNA Synthesis Kit (K1622), Pierce™ ECL western blotting substrate (32106), Pierce protein assay kit (23225), TRIzol^® reagent (15596‐018), CellROX™ Deep Red reagent (C10422), MitoSOX™ Red mitochondria superoxide indicator (M36008), and Pierce Renilla‐Firefly Luciferase Dual Assay Kit (16185) were purchased from Thermo Fisher Scientific (Waltham, MA). Anti‐GAPDH–HRP (HRP‐60004) was from ProSci (Fort Collins, CO). Mouse TGF Beta 1 PicoKine™ ELISA Kit was from BosterBio (Pleasanton, CA).