Patients’ adipose tissues and lipoma tissues were minced into small pieces and transferred into a Celstir spinner flask (Wheaton) supplemented with Serum-free Dulbecco’s modified eagle medium/f12 (DMEM/F12) and 1% penicillin/streptomycin, respectively. The tissues were cultured at 37 °C and a rotational speed of 100 rpm for 2 days. The debris of tissues and cells were removed by centrifugation (2000g, 30 min). An additional centrifugation in Amicon® Ultra-50 Centrifugal Filter Units with Ultracel-3 membrane (3000Mw cutoff membrane, Millipore) at 5000g for 30 min was applied to concentrate lipoma tissue extract (LTE) and adipose tissue extract (ATE). Then, the ATE and LTE were mixed with the Total Exosome Isolation™ reagent (Life Technologies) at 4 °C overnight and a final ultracentrifugation step was performed at 10,000g for 1 h at 4 °C. The obtained pellet was resuspended in 400 μl of PBS and stored at − 80 °C with known concentration determined by using the Pierce BCA protein assay kit (KeyGEN, China).
Pierce bca protein assay kit
Manufactured by Keygen Biotech
Sourced in China
The Pierce BCA Protein Assay Kit is a colorimetric detection and quantification method for determining the total protein concentration in a sample. It utilizes the bicinchoninic acid (BCA) reaction to measure the reduction of copper ions by proteins in an alkaline environment, producing a purple-colored complex that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Amicon ultra 50 centrifugal filter units with ultracel 3 membrane 3000mw cutoff membrane, by Merck Group (2 mentions)
Total exosome isolation reagent, by Thermo Fisher Scientific (2 mentions)
Immobilon western hrp substrate, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by GenStar (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Anti irf3, by Cell Signaling Technology (1 mentions)
Anti sting, by Proteintech (1 mentions)
Anti phospho nf kb p65 ser536, by Cell Signaling Technology (1 mentions)
Anti cd63, by ABclonal (1 mentions)
Anti alix 12422 1 ap, by Proteintech (1 mentions)
Anti cd81 66866 1 ig, by Proteintech (1 mentions)
Anti phospho sting ser366, by Cell Signaling Technology (1 mentions)
Anti phospho sting ser365, by Cell Signaling Technology (1 mentions)
Anti nf kb p65, by ABclonal (1 mentions)
Anti phospho irf3 ser396, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Beyotime (1 mentions)
Anti calnexin, by Abcam (1 mentions)
Protease inhibitor cocktail, by Keygen Biotech (1 mentions)
5 protocols using pierce bca protein assay kit
1
Adipose and Lipoma Tissue Extraction
2
Western Blot Analysis of MARCO Expression
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The antibodies used were as follows: rabbit-anti-human MARCO (Abcam, MA, USA) as primary antibody and anti-rabbit-IgG (H+L) (CST, MA, US) as secondary antibody.Western blot analyses were performed as below. In brief, total protein was isolated from tissue samples using RIPA buffer (CST, MA, US) with PMSF (GenStar, Beijing, China) and phosphatase inhibitor tablets (CWBIO, Jiangsu, China) and quantified using a Pierce BCA Protein Assay Kit (KeyGEN BioTECH, Jiangsu, China). The total protein samples were loaded and separated on SDS-PAGE gels and transferred to PVDF membranes (Roche, Basel, Switzerland). The membranes were blocked with 5% skim milk and incubated with the indicated primary antibodies overnight at 4°C, which was followed by incubation with the corresponding secondary antibodies for 2 h at room temperature. Signals were visualized by Immobilon Western HRP Substrate (Millipore, MA, USA) and captured by a ChemiDocTM Touch Imaging System (Bio-Rad, MA, USA). GAPDH was used as a loading control.
3
Isolation and Characterization of Adipose and Lipoma Tissue-Derived Extracellates
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Patients' adipose tissues and lipoma tissues were rst minced into small pieces and transferred into a Celstir spinner ask (Wheaton) supplemented with DMEM/F12 and 1% penicillin/streptomycin, respectively. The tissues were cultured at 37 °C with speed at 100 rpm for 2 days. The debris of tissues and cells were removed by centrifugation (2000 g, 30 min). An additional centrifugation in Amicon® Ultra-50 Centrifugal Filter Units with Ultracel-3 membrane (3000Mw cutoff membrane, Millipore) at 5000 g for 30 min was applied to concentrate lipoma tissue extract (LTE) and adipose tissue extract (ATE). Then, the ATE and LTE were mixed with the Total Exosome Isolation™ reagent (Life Technologies) at 4 °C overnight and a nal ultracentrifugation step was performed at 10000 g for 1 h at 4 °C. The obtained pellet was resuspended in 400 µl of PBS and stored at -80 °C with known concentration determined by using the Pierce BCA protein assay kit (KeyGEN, China).
4
Proteomic Analysis of Colon Tissue Fractions
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Colon tissues, cells, and EVs fractions were homogenized using the RIPA buffer (Jiangsu KeyGEN BioTECH, Nanjing, China), phosphatase inhibitor cocktail, and protease inhibitor cocktail (Jiangsu KeyGEN BioTECH, Nanjing, China). Pierce BCA Protein Assay Kit (Jiangsu KeyGEN BioTECH, Nanjing, China) was used to determine the protein concentration. Anti-CD63 (A5271, ABclonal), anti-Alix (12422-1-AP, Proteintech), anti-CD81 (66866-1-Ig, Proteintech), anti-Calnexin (ab22595, Abcam), anti-STING (19851-1-AP, Proteintech), anti-Phospho-STING (Ser366) (85735, Cell Signaling Technology), anti-Phospho-STING (Ser365) (72971, Cell Signaling Technology), anti-IRF3 (4302, Cell Signaling Technology), anti-Phospho-IRF3 (Ser396) (29047, Cell Signaling Technology), anti-NF-kB p65 (A19653, ABclonal), anti-Phospho-NF-kB p65 (Ser536) (3303, Cell Signaling Technology), anti-Beta-Actin (4970, Cell Signaling Technology), anti-GAPDH (AF1186, Beyotime Biotechnology) and secondary antibodies (KGP1201, Jiangsu KeyGEN BioTECH, Nanjing, China) were used for western blot.
5
Placental Protein Extraction and Western Blot Analysis
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Briefly, 10 mg placenta were ground in liquid nitrogen and transferred into EP tubes. Then, radioimmunoprecipitation assay lysate buffer was added to the tissues, which were homogenized on ice using an Ultrasonic Cell Disruptor and then centrifuged at 16 000 rpm for 30 min at 4°C. The protein concentrations were measured using the Pierce BCA Protein Assay Kit (Key-Gen Biotech, Nanjing, China). Next, the membranes were incubated with diluted primary antibody (ANP, 1: 150; Abcam, San Francisco, CA; NPR-A, 1: 200; Abcam, San Francisco, CA).
Subsequently, the membranes were incubated with secondary antibody (goat anti-rabbit, 1: 1,000; Zhongshan Biotechnology, Beijing, China) and then developed using an ECL Western Blotting Detection System. Band intensities were calculated using the AlphaEaseFC FluorChem 8900 software (Alpha Innotech Corporation, San Leandro, CA).
Subsequently, the membranes were incubated with secondary antibody (goat anti-rabbit, 1: 1,000; Zhongshan Biotechnology, Beijing, China) and then developed using an ECL Western Blotting Detection System. Band intensities were calculated using the AlphaEaseFC FluorChem 8900 software (Alpha Innotech Corporation, San Leandro, CA).
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