Pierce bca protein assay kit

Cells were detached with TrypLE (Thermo Fisher Scientific #12,604,039), washed with PBS 1X, and pelleted for 5 min at 1000 RPM. For nuclear proteins of Fig. 3D, nuclei were extracted by resuspending the cell pellet in buffer A (10 mM HEPES pH 7.65, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, 1X cOmplete Mini EDTA-free protease inhibitors (Roche)) then incubated for 15 min at 4 °C under gentle rotation. Nuclei were released from cells with the Dounce homogenizer, and pelleted at 250 g for 5 min at 4 °C. Nuclei were washed in buffer N (15 mM HEPES pH 7.65, 10 mM MgCl₂, 0.5 mM DTT, 250 mM sucrose, 1X cOmplete Mini EDTA-free protease inhibitors (Roche)) and pelleted at 2,800 g for 10 min at 4 °C. For nuclear proteins (Additional file 1: Fig. S2D), nuclei were extracted using the Subcellular Protein Fractionation Kit for Cultured Cells #78,840.
Cells or nuclei were lysed by resuspending the pellets in RIPA Buffer (150 mM NaCl, 1% IGEPAL, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) containing 1X cOmplete Mini EDTA-free protease inhibitors (Roche) and incubated 5 min on ice. Genomic DNA was digested using Universal Pierce Nuclease 250 U (Thermo Fisher Scientific #88,702) at 37 °C for 5 min and insoluble chromatin was removed by centrifugation for 1 min at 4 °C at 18,000 g. Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific #23,227). Equal amounts of proteins were loaded on 4–20% Tris–Glycine gel (NuPAGE), or 4–12% Bis–Tris gel (NuPAGE) for protein detection. Protein dry transfer was performed on 0.2 μm nitrocellulose membranes (BioRad #1,704,159) using a Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked for 1 h in Saturating Buffer (5% BSA, 0.1% Tween-20, PBS 1X) and incubated overnight at 4 °C in primary Antibody Buffer (5% BSA, 0.1% Tween-20, PBS 1X) with primary antibodies (Additional file 5: Table S4). The membranes were washed three times for 5 min at room temperature (RT) in Washing Buffer I (0.5% Triton X-100, 0.5 M NaCl, PBS 1X), one time for 10 min at RT in Washing Buffer II (0.5 M NaCl, PBS 1X), one time for 15 min at RT in PBS 1X and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Membranes were washed again as previously described and signal was revealed using the ECL-Prime Western Blot System (Sigma #RPN2232) on an Amersham ImageQuant 800 system.

Human liposuction aspirates were obtained from six healthy female donors who underwent liposuction from October 2017 to April 2018 in Shanghai 9th People’s Hospital, Shanghai, China after providing written informed consent. The mean age was 31 years (range 24–36 years). The study was approved by the Ethics Committee of Shanghai Jiaotong University School of Medicine, Shanghai, China. A standard traditional liposuction cannula with large side holes (2 mm × 7 mm) was used to harvest the macrofat, as previously described [20 (link)].
The detailed procedures for isolating FE are shown in Fig. 1. The lipoaspirate was first rinsed with saline to remove red blood cells and then centrifuged at 1200 × g for 3 min. After the first spin, the superior oily and inferior fluid layers were discarded, and the middle fat layer was collected and mechanically emulsified. The emulsification was achieved via 30 passes of shifting the fat between two 10-cm³ syringes connected by a female-to-female Luer-Lok connector (B. Braun Medical Inc., Melsungen, Germany). The emulsified fat was then frozen at − 80 °C and thawed at 37 °C for further disruption of the fat tissue. After one cycle of the freeze/thaw process, the fat was again centrifuged at 1200 × g for 5 min. After a second spin, the fat was separated into four layers. The upper layer of oil was discarded; the second layer of unbroken fat and the fourth layer of debris was discarded; and the third aqueous layer, namely the FE, was carefully aspirated without contamination of the bottom pellet. The final extract was produced by passing it through a 0.22-μm filter (Corning Glass Works, Corning, NY, USA) for sterilization and removal of cell debris. The extract was then stored at − 20 °C for future use. The protein concentrations of FE were measured with a Pierce BCA protein assay kit (Thermofisher Scientific, Waltham, MA, USA).Fig. 1

Schematic illustration of FE preparation

For protein extraction mid-jejunum mucosa samples were thawed on ice and 1 g of the sample was placed in sterile tube (5 mL tube, Eppendorf, Hamburg, Germany) followed by addition of 2 mL of PBS (MP Biomedicals, Inc., Santa Ana, CA, USA). Samples were homogenized (Tissuemiser, Thermo Fisher Scientific, Waltham, MA, USA) for 30 s and centrifuged at 87,000× g for 20 min on ice. The supernatant was subdivided into vials stored at −80 °C until being used to evaluate antioxidant status, immune response, and intestinal barrier function in mid-jejunum mucosa relative to the protein content of samples (PierceTM BCA Protein Assay Kit, Thermo Fisher Scientific, Waltham, MA, USA). Protein quantification started by mixing 25 µL of each sample with 200 µL of working reagent provided in the kit in a microplate well (96-Well EIA/RIA Plates, Corning, Corning, NY, USA), followed by 30 s incubation in plate shaker. The plate was covered with clear adhesive strip and incubated for 30 min at 37 °C. The plate was cooled to room temperature and wells were read at 562 nm.
The quantification of protein carbonyls (STA-310, Cell Biolabs, Inc., San Diego, CA, USA) as an index of oxidized proteins is described by Shen et al. [89 (link)]. Briefly, the protein content of each sample determined in the previous assay was diluted to 10 µg/mL. Diluted samples (100 µL) were pipetted into wells and incubated for 2 h at 37 °C. Each well was washed three times with 250 µL of PBS (MP Biomedicals, Inc., Santa Ana, CA, USA) and 100 µL of working solution supplied in the kit added before plate incubation in the dark for 45 min. Each well was washed with 250 µL of PBS/ethanol (1:1, v/v) and incubated for 5 min in an orbital shaker; this procedure was repeated four times. Each well was washed with 250 µL of PBS twice, 200 µL of blocking solution was added, and the plate was incubated for 1 h in an orbital shaker. Each well was washed with 250 µL of washing buffer three times and 100 µL of anti-dinitrophenylhydrazine antibody supplied in the kit were added according to dilutions recommended by the manufacturer. The plate was incubated in an orbital shaker for 1 h. Each well was washed with 250 µL of washing buffer three times and 100 µL of horseradish peroxidase antibody were added for incubation for 1 h in an orbital shaker. Each well was washed with 250 µL of washing buffer five times, 100 µL of substrate were added, and 100 µL of stop solution were added after the onset color development. The wells were read at 450 nm.
Malondialdehydes (STA-330, Cell Biolabs, Inc., San Diego, CA, USA) were measured by incubating for 5 min 100 µL of each sample in equal volume of SDS lysis solution provided in the kit. Followed by incubation at 95 °C for 45 min with 250 µL of the reagent (130 mg of thiobarbituric acid in 25 mL of diluent) supplied in the kit, which had the pH adjusted (Accumet AB15 pH Meter, Fisher Scientific, Hampton, NH, USA) to 3.5 with sodium hydroxide. Tubes were cooled in for 5 min and centrifuged at 4000× g for 15 min. The supernatant (300 µL) was vigorously mixed with 300 µL of butanol for 2 min and centrifuged at 10,000× g for 5 min. The supernatant (200 µL) was transferred to a microplate (96-Well EIA/RIA Plates, Corning, Corning, NY, USA) and samples were read at 532 nm.
Tumor necrosis factor-α (PTA00, R&D Systems, Inc., Minneapolis, MN, USA) was measured by pipetting 50 µL of assay diluent supplied in the kit with 50 µL of samples into wells. The plate was covered with clear adhesive strip and incubated for 2 h. Each well was washed five times with 300 µL of washing buffer, 100 µL of TNF-α conjugate supplied in the kit were added, and the plate was incubated following same specifications. Each well was washed five times with 300 µL of washing buffer, 100 µL of substrate solution supplied in the kit were added to each well, and the plate was incubated for 30 min in the dark. After incubation, 100 µL of stop solution supplied in the kit were added and wells were read 450 and 570 nm to obtain reading at 570 subtracted from 450 nm.
Iterleukin-8 quantification (P8000, R&D Systems, Inc., Minneapolis, MN, USA) was performed by pipetting 50 µL of assay diluent supplied in the kit with 100 µL of samples into wells. The plate was covered with clear adhesive strip and incubated for 2 h in orbital shaker at 500 rpm. Each well was washed five times with 300 µL of washing buffer, 200 µL of porcine IL-8 conjugate supplied in the kit were added, and the plate was incubated following same specifications. Each well was washed five times with 300 µL of washing buffer, 120 µL of substrate solution supplied in the kit were added, and the plate incubated for 30 min in the dark. After incubation, 120 µL of stop solution supplied in the kit were added and wells were read 450 and 570 nm to obtain reading at 570 subtracted from 450 nm.
Immunoglobulin A (E100-102, Bethyl Laboratories, Inc., Montgomery, TX, USA) and IgG (E100-104, Bethyl Laboratories, Inc., Montgomery, TX, USA) were measured by pipetting 100 µL of their respective affinity purified antibody in each well according to the kit dilution. The plate was incubated for 1 h. Each well was washed five times with 260 µL of washing buffer supplied in the kit, 200 µL of blocking buffer supplied in the kit were added, and the plate was incubated for 30 min. Each well was washed five times with 260 µL of washing buffer, 100 µL of samples were added and incubated for 30 min. Each well was washed five times with 260 µL of washing buffer, 100 µL of diluted horseradish peroxidase supplied in the kit were added, and the plate was incubated for 1 h. Each well was washed five times with 260 µL of washing buffer, 100 µL of tetramethylbenzidine substrate were added, and the plate was incubated in the dark for 15 min. Sulfuric acid (100 µL) at 0.18 M was used as stop solution. The plate was read at 450 nm.
For measurement of total glutathione, a different protein extraction method was used, as recommended by the kit manufacturer total glutathione (STA-312, Cell Biolabs, Inc., San Diego, CA, USA). Mid-jejunum mucosa (100 mg) and 1 mL of metaphosphoric acid at 5% were mixed and homogenized with a glass pestle. The homogenate was centrifuged at 64,000× g for 15 min. The supernatant was used for total glutathione determination total glutathione (STA-312, Cell Biolabs, Inc., San Diego, CA, USA). Glutathione reductase solution (25 µL), NADPH solution (25 µL) supplied in the kit, and samples (100 µL) were added to each well. The chromogen solution (100 µL) supplied in the kit was added to each well and the plate was read at 405 nm every 2 min during 10 min. All wavelengths (for quantifications of protein, protein carbonyls, malondialdehydes, total glutathione, TNF-α, IL-8, IgA, and IgG) were read at the same microplate reader (Synergy HT, Biotek, Winooski, VT, USA).
Ileal digesta was freeze dried (SP Scientific, Virtis 24DX48 GPFD/300820, Warminster, PA, USA) and ground. Subsamples of ground material were analyzed for apparent ileal digestibility of dry matter [90 (link)], gross energy (6200 Calorimeter, Parr Instrument Company, Moline, IL, USA), nitrogen (method 990.03, [91 ], ATC Scientific, North Little Rock, AR, USA), and ether extract (method 920.39, [91 ]).
Fixed mid-jejunal tissue was removed from 10% buffered formaldehyde after two weeks for the obtainment of two transversal cuts that were transferred histological cassettes and submerged in 70% ethanol. Mid-jejunal cuts were included in paraffin for assembling histological slides after staining for Ki-67 antigen. The immunohistochemistry staining with Ki-67 primary monoclonal antibody (1:500 dilution) followed by anti-mouse secondary antibody (1:2 dilution factor) and the use of diamino-benzamine reagent for color development was performed in accordance with methods previously described by Kim et. al. [20 (link)]. Ten pictures of each pig were used to measure gut morphology by a single researcher choosing a well-oriented villus and its associated crypt. Measurements included: villus width (at half of villus height), villus height (from tip of the villus to top of the crypt), crypt depth (from top to bottom of the crypt), and calculating villus height: crypt depth [86 (link)]. The proportion of proliferating cells in the crypt was also estimated by calculating the proportion of cells positive to Ki-67 after taking pictures at 40× in Sony Van–Ox S microscope (Opelco, Washington, DC, USA) and processing in ImageJS tool [92 (link)] for analysis as described by Holanda and Kim [86 (link)].