Remaining splenocytes were used to isolate proteins for WB analysis. Protein extraction was performed according to the manufacturer’s instructions of a commercially-available protein extraction kit (Cell Extraction Buffer, Invitrogen, Vienna, Austria). Cells were washed with phosphate buffered saline (PBS) and then resuspended in the respective amount of Cell Extraction Buffer (1 ml/108 cells; Life Technologies GmbH, Darmstadt, Germany) supplemented with complete mini protease inhibitor (Roche Diagnostics GmbH, Mannheim, Germany). After 30 min of incubation on ice, cell suspensions were centrifuged and the supernatants were collected and stored at -20°C until analysis. The protein concentration was determined using a commercially-available quantification kit (Bicinchoninic Acid Protein Assay Kit, Thermo Scientific, Rockford, USA).
Cell extraction buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Switzerland, Austria, United Kingdom, Belgium, China
Cell extraction buffer is a solution used to extract proteins and other cellular components from biological samples, such as cells or tissues. It helps to disrupt cell membranes and release the contents of the cells, allowing for further analysis or purification of the desired components.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (42 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (15 mentions)
Na3vo4, by Merck Group (9 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (8 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Gapdh, by Cell Signaling Technology (7 mentions)
Halt protease and phosphatase inhibitor, by Thermo Fisher Scientific (7 mentions)
Protease inhibitor cocktail, by Roche (6 mentions)
Protease inhibitor, by Merck Group (6 mentions)
Anti rabbit secondary antibody, by Cell Signaling Technology (6 mentions)
Quantity one 4, by Bio-Rad (6 mentions)
Dc protein assay, by Thermo Fisher Scientific (6 mentions)
Whatman, by Cytiva (6 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (5 mentions)
Criterion 4 to 12 bis tris gels, by Bio-Rad (5 mentions)
Odyssey fc, by LI COR (5 mentions)
Criterion blotter wet transfer, by Bio-Rad (5 mentions)
Polyvinylidene difluoride membrane, by Merck Group (4 mentions)
222 protocols using cell extraction buffer
1
Protein Extraction and Quantification
2
Brain Tissue Homogenization for Protein Extraction
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Fresh frozen left-brain hemispheres were homogenized in cell extraction buffer (Invitrogen) using the following protocol: frozen brain was transferred to a Dounce tissue homogenizer, after which 1 mL of cell extraction buffer (Invitrogen, Carlsbad, California) was added. Brain tissue was mechanically homogenized, followed by incubation on ice for 30 min. Every 10 min, the homogenate was vortexed and resuspended. Subsequently, the samples were centrifuged for 30 min at 13,000×g and 4°C. The supernatant was collected in a fresh Eppendorf tube, and both supernatant and pellet were stored at −20°C until further use.
3
Lutein Modulates MAPK and NF-κB in CE Cells
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CE cells at a density of 1 × 106 cells/well were seeded on 6-well plates and cultured for 24 h. Then, culture medium was removed and washed and cells were cultured with isoosmotic or hyperosmotic medium with and without lutein (10 μM) as described above. The cultures were washed with cold PBS 60 min later. Cells were collected and centrifuged. The pellets were cultured with cell extraction buffer (Biosource, Camarillo, CA, USA), protease inhibitor cocktail (Sigma), and PMSF (Biosource) for 30 min at 4°C with vortexing at 10 min intervals. Cultures were microcentrifuged at 4°C and the supernatants were collected and stored at −80°C until analysis for mitogen-activated protein kinase (MAPK) levels. For the measurement of nuclear factor-kappa B (NF-κB) levels in cell nucleus, cells were collected, treated with hypotonic buffer (BioSource), and centrifuged. The pellets that contained nuclear fraction were collected, treated with cell extraction buffer (BioSource), vortexed, and centrifuged. The supernatants were stored at −80°C until analysis for NF-κB levels.
4
Isolation of Cytoplasmic and Nuclear Fractions from NSCLC Cells
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The 5 × 106 human NSCLC cells were suspended in hypotonic buffer (20 mM Tris-Cl pH 7.4, 10 mM NaCl, 3 mM MgCl2, protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (PMSF) Cell Signaling Technology) and incubated on ice for 15 min. Nonionic detergent NP-40 (10%; Sigma-Aldrich) was then added to the cell suspension, which was mixed vigorously. Next, the cell homogenate was centrifuged at 5000 rpm for 10 min at 4 °C. The supernatant was collected as the cytoplasmic fraction, and the pellet was suspended in cell extraction buffer (Thermo Fisher Scientific) supplemented with protease inhibitor cocktail (Roche) and 1 mM PMSF. The suspension was incubated on ice for 30 min with intermittent vortexing. Finally, the sample was centrifuged at 14,000 × g for 30 min at 4 °C, and the supernatant was collected as nuclear extract.
5
Western Blot Analysis of IEC Innate Immune Signaling
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Total cell lysates of IECs transfected with Poly I:C was prepared by using the cell extraction buffer (Thermo Fisher Scientific, MA, USA) according to the manufacturer’s instructions. Equal amounts of protein lysates (30 µg) were separated on 4–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis precast gels and transfected to an Immunobiolon-P membrane (Millipore, Eschborn, Germany). The blots were incubated with primary antibodies in 5% nonfat milk in PBS with 0.05% Tween 20 (PBST) overnight at 4°C (IRF3, 1:1,000; Phospho-IRF3, 1:1,000; IRF7, 1:1,000; Phospho-IRF7, 1:1,000; GAPDH, 1:5,000; β-actin, 1:5,000; EEA1, 1:1,000; CD63, 1:1,000; LAMP2, 1:2,000; Alix, 1:1,000; ISG15, 1:1,000; ISG56, 1:1,000; GBP5, 1:1,000; Viperin, 1:1,000; MxA, 1:1,000; MxB, 1:1,000; OAS-1, 1:1,000). All antibodies were obtained from Cell Signaling Technology (Cell Signaling Technology, MA, USA) Horseradish peroxidase-conjugated appropriate second antibodies were diluted at 1:2,000 to 1:8,000 in 2% nonfat milk PBST. Blots were developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, MA, USA).
6
SARS-CoV-2 Virus Inactivation Protocol
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All work with infectious SARS-CoV-2, isolate USA_WA1/2020 was performed at the National Emerging Infectious Diseases Laboratories, Boston University under Biosafety Level 4. Vero E6 cells seeded in 6-well plates were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 1 or mock infected. After 1 h of virus adsorption at 37°C in 5% CO2, the inocula were removed and replaced with DMEM supplemented with 2% FBS. Twenty-four hours post-infection, cell supernatants were removed, the cells were scraped and pelleted by low-speed centrifugation. Cell pellets were washed once with PBS and resuspended in 270 μl Cell Extraction Buffer (Thermo Fisher Scientific). A 900 μl aliquot of a SARS-CoV-2 stock (Titer: 1.58 ×107 TCID50 units/ml) was also used for analysis. Both the cell lysates and the viral stock were inactivated by addition of SDS (1 % final concentration) and boiling for 10 minutes prior to removal from the Biosafety Level 4 facility. The inactivated cell lysates and virus stock were aliquoted and stored at −80°C until further use. The protein concentration was quantified by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific).
7
Quantifying IL-10 in BM-MSCs and Infarcted Hearts
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BM-MSCs were seeded in 12-well plate with 10 × 106 cell/well. 48 h later, cell culture supernatant was collected and the IL-10 level was determined using commercial ELISA kit (R&D systems, Minneapolis, MN, USA) following manufactures’ protocol. In certain experiments, the peri-infarct area in the hearts was isolated and homogenized in cell extraction buffer (Thermo Fisher) following manufactures’ protocol. The IL-10 level in homogenates was measured using ELISA kit.
8
Testicular Cytokine Quantification
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Testicular sample (100 mg) was homogenized in ice-cold cell extraction buffer (Thermo Fisher Scientific, Waltham, MA, USA) with protease inhibitors (Sigma Chemical Company). After homogenization, the homogenate was centrifuged at 14,000 × gravity at 4°C for 15 minutes to obtain supernatant. The TNF-α and IL-1β levels in the supernatant were determined by using commercially available ELISA kits (PeproTech, Cranbury, NJ, USA) according to the manufacturer's instructions. The results of TNF-α and IL-1β levels were expressed as pg/mg tissue.
9
Western Blot Protein Expression Analysis
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Protein expression was confirmed using Western blotting. Cells were lysed using Cell Extraction Buffer (Thermo Fisher, Waltham, MA, USA) with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher). Concentrations of whole-cell lysate were measured using a BCA protein assay kit (Thermo Fisher). Whole cell lysates were electrophoresed on SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). The membrane was blocked treating with 3% bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween 20 (TBST) for 2 h at 25℃. The membranes were allowed to react overnight at 4℃ with primary antibodies and rinsed three times for 15 min each with TBST. Subsequently, they were reacted with horseradish peroxidase-conjugated secondary antibody for 1 h at 25℃ and rinsed with TBST for times for 15 min each. Protein expression was activated using ECL solution (WESTSAVE ECL Solution, AbFrontier, Seoul, Korea) and exposed to X-ray film (AGFA, Mortsel, Belgium). Information on the antibodies used is summarized in Table 1 .
10
Amyloid-Beta Protein Quantification
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After 24 h incubation, the medium in the cell culture flasks was collected and the cells were rinsed with cold PBS. The suspension was then centrifuged at 220× g for 5 min. Then, the supernatant was discarded and the cell pellet was resuspended in 1.5 mL PBS and protease inhibitor cocktail (Merck, Darmstadt, Germany). The suspension was then centrifuged at 112× g for 5 min and the supernatant was removed. The cell pellet was collected and 600 µL of cell extraction buffer (Thermo Fisher Scientific, Waltham, MA, USA) was added and incubated for 30 min. After that, the suspension was centrifuged at 13,000× g for 10 min. The supernatant was transferred to a new tube and stored at −80 °C. The supernatant was thawed on ice and pipetted onto a 384-well plate (Greiner Bio-One, Kremsmuenster, Austria). To measure the amyloid β-protein concentration, an HTRF-Amyloid-Beta 1-40 Kit (Cisbio, Codolet, France) was used, with samples treated according to the manufacturer’s instructions. The optical density was then measured at 665 and 622 nm emission wavelengths. Aβ levels were normalized against protein content.
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