Bicinchoninic acid bca kit

The study used the following reagents: paraquat (Sigma-Aldrich, United States); metformin (Sigma-Aldrich, United States); hematoxylin and eosin (HE) staining reagent (Department of Pathology of Zhengzhou University, China); Rat interleukin (IL)-6 (No.70-EK306/3-96), tumor necrosis factor (TNF)-α (No.70-EK382HS-96), and IL-10 (No.70-EK310/2-96); enzyme-linked immunosorbent assay (ELISA) kits (Multi Sciences (LIANKE) Biotechnology Company, China); fetal bovine serum (Gibco, United States); F12K culture medium (Sigma-Aldrich, United States); cell counting kit (CCK-8) assays (Dojindo, Japan); bicinchoninic acid (BCA) kit (Thermo Fisher Scientific, United States); cell lysis solution (GBCBIO, Guangzhou, China); quantitative polymerase chain reaction (q-PCR) kit (TaKaRa, Dalian, China); polyvinylidene fluoride membrane (Millipore, Bedford, MA); inducible nitric oxide synthase (iNOS) (SC-7271) and arginase 1 (Arg1) (SC-271430) (Santa Cruz Biotechnology, United States); and β-actin (13E5) (Cell Signaling Technology, United States).

Western blotting was performed to examine the phosphorylation of p50, p65, IκB (NF‐κB pathway), Akt (AKT pathway), p38, ERK, C‐fos and JNK (MAPK pathway) in RAW264.7 cells. Cells induced by M‐CSF (30 ng/mL) and RANKL (50 ng/mL) with or without guaiacol (0 and 1.0 μmol/L) were incubated in a 96‐well plate for 7 days. Next, the expression levels of osteoclastogenesis‐related genes (encoding cathepsin K, CTR, MMP‐9 and TRAP) were assayed. Proteins were prepared and quantified using a bicinchoninic acid (BCA) kit (Thermo Fisher), resolved by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, electrotransferred onto a membrane, and blocked in Tris‐buffered saline with Tween in 5% skim milk. After incubation with the primary antibodies overnight (4°C), the samples were incubated with anti‐rabbit horseradish peroxidase‐conjugated secondary antibodies. The results were visualized by chemiluminescence (Bio‐Rad). All experiments were conducted for 3 times, and the average was calculated.

Dulbecco’s modified Eagle’s medium (DMEM) containing fetal bovine serum (FBS), penicillin‒streptomycin, and actinomycin D was purchased from Sigma-Aldrich (USA). TRIzol reagent, Lipofectamine 3000, anti-insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) primary antibody, bicinchoninic acid (BCA) kit, electrochemiluminescence (ECL) kit, BODIPY-C11, and SYBR Premix Ex Taq were purchased from Thermo Fisher Scientific (USA). The CCK-8 reagent was purchased from Dojindo Laboratories (Kumamoto, Japan). EdU reagent and RIPA lysis buffer were purchased from Beyotime (Shanghai, China). Anti-solute carrier family 7 member 11 (SLC7A11), anti-m6A, anti-U2AF2, anti-IgG antibodies, and an iron assay kit were purchased from Abcam (Shanghai, China). A fluorescence in situ hybridization (FISH) kit was purchased from RiboBio (Guangzhou, China). The Magna MeRIP m6A kit was purchased from Millipore (USA). The Magnetic RNA Protein Pull-Down Kit was purchased from Pierce (USA).

Nanoparticle particle size analysis was as follows: 20 μg EVs were dissolved in 1 mL PBS and mixed for 1 min for homogenous distribution. The NTA tool (Malvern, UK) was adopted for the particle size distribution of EVs.
TEM observation was as follows: 20 μL of ultra-isolated fresh EVs was loaded onto a carbon-coated copper electron microscope grid for 2 min, and then dyed in phosphotungstate solution (12501-23-4, Sigma-Aldrich) for 5 min. The grid was rinsed by PBS to eliminate any excess phosphotungstate solution, and semi-dried. The observation image was captured at 80 KV under a Hitachi H7650 TEM (Japan).
Western blot analysis was conducted for the identification of the EV-surface markers. After the concentration of EV suspension, the bicinchoninic acid (BCA) kit (23227, Thermo Fisher Scientific, San Jose, CA, USA) was utilized for protein concentration measurement. Next, a regimen of sodium dodecyl sulfate-polyacrylamide gel was prepared and the protein denaturation and electrophoresis were performed. Subsequently, the proteins on gels were transferred onto the membrane for analysis of the EV-specific proteins TSG101 (ab125011), CD9 (ab263019), Alix (ab388388), and Calnexin (ab22595) with antibodies from Abcam.

The size and surface zeta potential of the nanoparticles were determined by dynamic light scattering (DLS, Malvern ZEN 3600 Zetasizer). For examining the morphology with transmission electron microscopy (TEM), the nanoparticles solution (10 μg mL⁻¹) was dropped on a carbon‐coated copper grid (400‐mesh, Electron Microscopy Sciences), washed with DI water, and stained with uranyl acetate (1 wt%, Sigma‐Aldrich). The grid was imaged with an FEI 200KV Sphera microscope. The peptide content on the ctLP‐NPs was quantified using a bicinchoninic acid (BCA) kit (Thermo Fisher Scientific) in reference to a bovine serum albumin (BSA) standard. The stability of bare PLGA cores, LP‐NPs, and ctLP‐NPs were measured by monitoring their sizes in 1× PBS over a period of 1 week.

All of the chemicals used in the present study were analytical grade reagents from various sources. Phosphate-buffered saline (PBS), formaldehyde acetic acid and ammonium hydroxide, aminoguanidine bicarbonate (AG), MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and FPS-ZM1 were purchased from Merck Spa (Milan, Italy). Laemmli buffer and 2′–7′-dichlorofluorescein-diacetate (H₂DCF-DA) were purchased by Thermo Fisher Scientific (Milan, Italy), Roti-Block from Prodotti Gianni (Milan, Italy), and bicinchoninic acid (BCA) kit from Thermo Fisher Scientific (Milan, Italy).

ESCC cell lines (KYSE150, KYSE270, KYSE450, TE4, and TE8) and a normal esophageal epithelial cell line (HET-1A) were obtained from ATCC (MA, USA). The Roswell Park Memorial Institute (RPMI)-1640 medium, 5-diphenyltetrazolium bromide (MTT) kit, and 4′,6-Diamidino-2-Phenylindole (DAPI) stain solution were supplied by Sigma (MO, USA). Lipofectamine 3000 reagents and Bicinchoninic Acid (BCA) kit were purchased from Thermo Scientific (CA, USA). Fetal Bovine Serum (FBS) was procured from GIBCO (CA, USA). Horseradish Peroxidase (HRP)-conjugated goat anti-rabbit/mouse antibodies were obtained from Cell Signaling Technology (MA, USA). Alexa Fluor 488 or 555 donkey anti-rabbit/mouse secondary antibodies were purchased from Beyotime (Shanghai, China). Information on primary antibodies used in this study is listed in Table S1.