Coomassie brilliant blue g 250 kit

The integrity of the bacterial cell membrane can be reflected by the leakage of intracellular compounds. The extracellular protein concentration of the bacterial suspension was determined by using the Coomassie Brilliant Blue G-250 kit (Beijing Solarbio Science & Technology Co., Ltd. Beijing, China). Briefly, Salmonella cells (10⁷ PFU/mL) in logarithmic phase were unexposed or exposed with 125 μg/mL MnO₂ nanosheets for 2 h at 4 °C, followed by centrifugation at 10,000 r/min for 5 min to collect the supernatant. Next, the treated bacterial solution (50 μL) was supplemented with 200 μL of the protein working solution in each well of the 96-well plate and then cultured at 37 °C for 5 min. Finally, the protein leakage was evaluated by measuring the absorbance at 562 nm using an Enzyme Linked Immunosorbent Assay (ELISA) microplate reader (Waltham, MA, USA).

Manganese chloride tetrahydrate (MnCl₂·4H₂O) and tetramethylammonium hydroxide were purchased from Shanghai Reagent Chemical Co. (Shanghai, China). Hydrogen peroxide (H₂O₂, 30 wt%) and other chemicals of analytical grade were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), trypsin, fetal bovine serum (FBS), MTT and penicillin streptomycin were obtained from GIBCO Invitrogen Corp (Waltham, MA, USA); 2′,7′-dichlorofluorescin diacetate (DCFH-DA) from Sigma-Aldrich (St. Louis, MO, USA); propidium iodide (PI), 4′-6-diamidino-2-phenylindole (DAPI) and Coomassie brilliant blue G-250 kit products from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).

According to the instruction of manufacturer, total protein was extracted from intestinal tissues and IEC-6 cells, respectively. The supernatant was collected and quantified using a Coomassie Brilliant Blue G-250 kit (Solarbio, Beijing, China). 10% SDS-PAGE gel was used to separate the protein samples (25 μg), and the separated samples were transferred to PVDF membrane. 5% skimmed milk or 5% bovine serum albumin was diluted in Tris-buffered saline containing Tween (TBS-T) to seal the membranes (3 h, 37°C). And the sealed membranes were hatched overnight (4°C) with following primary antibodies: Bcl-2-associated X (Bax), B-cell lymphoma-2 (Bcl-2), cysteinyl aspartate specific proteinase 3 (caspase-3), SOD-2, Nrf2, heme oxygenase 1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1), and β-actin (Proteintech, Wuhan, China). After the first antibody was incubated, the imprints were rinsed in TBS-T for three times and then were hatched (room temperature, 2 h) with a horseradish-conjugated goat anti-rabbit antibody (Proteintech, Wuhan, China). ECL detection system was used to detect protein abundance. Image Lab software (Bio-Rad, CA, USA) was used to measure protein quantitation in optical density units and standardized to the corresponding β-actin sample expression.