Western blot analysis was used to determine the expression of proteins related to the cell cycle to confirm the effects of PBMCs on EMT markers and also to investigate the levels of H3K9 acetylation and phosphorylation of H2AX. Co-cultures of PBMCs and the different cell lines of keratinocytes were grown in 100 mm2 plates with 70% to 80% confluence and then washed with cold PBS, and subsequently, the cells were detached from the plates with a cell scraper (Costar, Washington, DC, USA). After centrifugation, the cell precipitates were incubated with 100 μL of lysis buffer (Roche, Grenzach-Wyhlen, Germany), and protein concentrations were measured using the Bradford method (Bio-Rad, Hercules, CA, USA). Standardized amounts of total protein for each antibody reaction were electrophoretically separated in a polyacrylamide gel with 10% sodium dodecyl sulfate (SDS-PAGE) under reducing conditions and transferred to nitrocellulose membranes (Millipore, MA, USA). The membranes were blocked with 5% fat-free milk and then incubated with primary antibodies (Table S2 ) followed by a secondary peroxidase-conjugated antibody. After washing, the protein bands were detected using an enhanced chemiluminescence (ECL) Western Blotting System (Bio-Rad Laboratories, Hercules, CA, USA).
Cell scraper
Manufactured by Corning
Sourced in United States, Mexico
The Cell Scraper is a laboratory tool used to detach and collect adherent cells from the surface of a cell culture dish or flask. It consists of a flat, flexible blade attached to a handle, designed to gently scrape and lift the cells without damaging their structure.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Cool lysate, by Beyotime (2 mentions)
Protein a agarose beads, by Beyotime (2 mentions)
Anti flag antibodies f1804, by Merck Group (2 mentions)
Page loading buffers, by Beyotime (2 mentions)
B900620, by Proteintech (2 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (2 mentions)
Stempro accutase, by Thermo Fisher Scientific (2 mentions)
Phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
1 thioglycerol, by Merck Group (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Ecl western blotting system, by Bio-Rad (1 mentions)
Lysis buffer, by Roche (1 mentions)
Bradford method, by Bio-Rad (1 mentions)
Reverse transcription kit, by Takara Bio (1 mentions)
54 protocols using cell scraper
1
Protein Expression Analysis of EMT and Chromatin Modifications
2
Establishing Primary Osteosarcoma Cell Line
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A 27 year-old male Chinese osteosarcoma patient received biopsy for diagnosis purpose before chemotherapy and surgery. The biopsy sample was washed with warm PBS and minced under sterile condition, followed by digestion with 150 U mL−1 (in DMEM complete culture medium, described later) of Type I collagenase for 3 hours under 37 °C. Single cells suspension was obtained by passing the biopsy sample after digestion through a 70 μm cell strainer (Sangon Biotech, Shanghai, China) and centrifugation. The cell pellet was resuspended in DMEM high glucose medium (Zhong Qiao Xin Zhou Biotechnology, Shanghai, China) supplemented with 10% FBS (Invitrogen) and 1% penicillin/streptomycin stock solution (Sciencell), and the cells were cultured in a 35 mm Petri dish in a humidified atmosphere at 37 °C with 5% CO2 until confluent without changing the medium. Cells were then sub-cultured at a 1 : 5 split ratio, and osteosarcoma cell colonies were picked out with a cell scraper (Costar). The osteosarcoma cells were maintained in vitro as described above, and their tumorigenicity was confirmed by tumor formation assay using 6 week-old male BALB/c nude mice (Vital river, Beijing, China).
3
Intracellular Metabolite Extraction Protocol
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Cell intracellular extracts were prepared following a direct quenching method described previously.23 (link) To minimize the influence of the carryover of culture medium, cells were quickly rinsed thrice by phosphate-buffered saline (PBS, pH 7.4). The residual PBS was removed immediately by vacuum suction. Subsequently, cells were quenched by using 3 mL of HPLC grade methanol before being gently detached from the culture dish using a cell scraper (Costar, Mexico). The methanol solution containing quenched cells was pipetted into a 15 mL centrifuge tube for extraction. A mixture of methanol, chloroform and water in the volume ratio of 4 : 4 : 2.85 was applied in a dual phase extraction for extracting intracellular metabolites. Standing for 30 min on the ice, the mixture was centrifuged at 12 000g for 15 min at 4 °C. Finally, only the aqueous phase was lyophilized and subjected to NMR-based metabolomic analysis.
The aqueous cell extract powder was resolved in 550 μL of phosphate buffer [50 mM, pH 7.4, 10% D2O, 0.01 mM sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TSP) and 0.02% NaN3], vortexed and then centrifuged at 12 000g for 15 min at 4 °C. Aliquots of the supernatant were transferred into 5 mm NMR tubes.
The aqueous cell extract powder was resolved in 550 μL of phosphate buffer [50 mM, pH 7.4, 10% D2O, 0.01 mM sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TSP) and 0.02% NaN3], vortexed and then centrifuged at 12 000g for 15 min at 4 °C. Aliquots of the supernatant were transferred into 5 mm NMR tubes.
4
FMRP-Bound RNA Isolation Protocol
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We cultured four dishes (10 cm) of HCCLM3 cells and harvested them with a cell scraper (Costar, USA) in 10 mL of ice-cold PBS, which were then collected by centrifugation and broken up with polysome lysis buffer. Protein A + G agarose (Millipore, USA) was incubated with 5 μl of rabbit anti-FMRP (Abcam, UK) or normal IgG (Beyotime, China) for 1 h. Cell lysates were stored at −80 °C overnight before being centrifuged. Then, the A + G agarose-antibody was incubated with the cell lysates at 4 °C overnight. The following day, the agarose was washed with 1 mL of NT2 buffer (supplemented with 1 M urea, 0.1% SDS, and 150 mM NaCl) six times. NT2 buffer supplemented with 180 µl of proteinase K was used to digest the proteins by incubating the tubes at 55 °C for 30 min with shaking. Then, 1 mL of TRIzol reagent was added for RNA extraction, after which 2 μl of glycogen (20 mg/ml) in isopropanol was added to precipitate the RNA. To avoid DNA contamination, we used reverse transcription kits (Takara, Japan), which are able to remove DNA.
5
NMR Analysis of C2C12 Metabolites
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Aqueous metabolites were extracted from C2C12 myoblast cells for NMR analyses according to the protocol described previously [41 (link)]. Cells were quickly rinsed thrice by cold phosphate-buffered saline (PBS, pH 7.4) to reduce the residual medium. The residual PBS was removed immediately by vacuum suction. Subsequently, metabolic activities of cells were aborted by methanol, and cells were scraped off by a cell scraper (Costar, Washington, DC, USA). Cells were then collected into a 15 mL centrifuge tube. Thereafter, methanol, chloroform, and water in the volume ratio of 4:4:2.85 were applied in a dual-phase extraction for extracting intracellular metabolites. Only the polar phase was lyophilized and subjected to metabolomic profiling. The aqueous cell extract powder was resolved in 550 μL of phosphate buffer [50 mm, pH 7.4, 100% D2O, 0.05 mm sodium 3-(trimethylsilyl) propionate-2,2,3,3-d4 (TSP)], vortexed and then centrifuged at 12,000× g for 15 min at 4 °C. The supernatants were transferred into 5 mm NMR tubes for NMR-based metabolomic analysis.
6
Generating Bone Marrow-Derived Macrophages
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Bone marrow-derived macrophage (BMDM) cells from mice were stimulated with recombinant mouse M-CSF (R&D) as reported previously21 (link). Briefly, the bone marrow cells were stimulated with 20 ng/ml M-CSF. After 7 days culture, more than 90% pure BMDM were obtained. For the following tests, BMDM cells were harvested using a cell scraper (Costar) and cultured at 5 × 105 cells/well in a fresh 24-well plates overnight.
7
Quantitative Determination of Cellular Enzyme Activities
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The enzymatic activities were detected using a GENMED Cellular PKA Activity Colorimetric Quantitative Determination Kit, a GENMED Cellular PI3K Activity Colorimetric Quantitative Determination Kit and a GENMED Cellular PKB/AKT Activity Colorimetric Quantitative Determination Kit (GENMED) according to the manufacturer's instructions. In brief, cells were cultured in T-75 flasks, removed carefully with a cell scraper (Corning Incorporated Life Sciences, Acton, MA, USA), pelleted by centrifugation at 3000 r.p.m. for 5 min, and lysed with 500 μl lysis buffer for 30 min on ice. Cell lysates were then centrifuged at 20 000 × g for 20 min at 4 °C. Total proteins in the supernatants were quantified with the BCA reagent (Beyotime). Protein (50 μg in 5 μl) was transferred to a 96-well plate in which each well contained 95 μl reaction mixture and incubated for 5 min at 37 °C. Samples were then read with a microplate reader (Bio-Rad).
8
Osteogenic Differentiation of hDPSCs in 2D and 3D
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hDPSCs were seeded into six-well culture plates (Falcon Corning) at a density of 1 × 105 cells/well and cultured in basal medium for 4 weeks. The medium was changed twice a week. At 4 weeks, DPSCs formed a thick cell sheet called the basal sheet. To prepare the hDPSC constructs, basal sheets were gently detached using a cell scraper (Falcon Corning) from the plates, and cultured for another week on ultra-low cell-binding six-well plates (HydroCell, Cell Seed Europe Ltd., London, UK) with basal or osteogenic medium that comprised basal medium supplemented with 50 μg/mL of ascorbic acid (Wako Pure Chemical Industries Ltd., Osaka, Japan), 10 mM of β-glycerophosphate (Sigma-Aldrich), and 10−8 M dexamethasone (Sigma-Aldrich) for 3D culture. The medium was changed twice a week. As a control, basal sheets were cultured for 1 week in the same six-well plates with basal and osteogenic media for monolayer culture (2D).
Four types of cultures were prepared and defined as follows:
Four types of cultures were prepared and defined as follows:
hDPSC sheet: cultured in basal medium under 2D culture.
o+hDPSC sheet: cultured in osteogenic medium under 2D culture.
hDPSC construct: cultured in basal medium under 3D culture.
o+hDPSC construct: cultured in osteogenic medium under 3D culture.
9
Quantification of γ-GT in BMEC Cultures
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BMECs in the co-culture model and single layer BMEC groups were washed three times in cold PBS, collected gently using a cell scraper (Corning), centrifuged at 700 × g or 5 minutes, and the supernatant was discarded. Cells in each well were lysed with 150 µL cell lysate (YuanYe Biological Technology Co., Ltd., Shanghai, China) on ice for 30 minutes (shaken every 10 minutes), and centrifuged at 1,104 × g or 30 minutes at 4°C. The filtrate was aliquoted and stored. γ-GT was measured using an enzyme linked immunosorbent assay kit (YuanYe Biological Technology Co., Ltd.) according to the manufacturer's instructions. The optical density of each well was measured with a microplate reader (Awareness Technology, Palm City, FL, USA) at a wavelength of 450 nm.
10
Flag-tagged Protein Immunoprecipitation
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The cells were collected by a cell scraper (Corning) with cool lysate (Beyotime) for protein extraction. The lysate was incubated with anti‐Flag antibodies (F1804; Merck, Darmstadt, Germany) or an equal amount of mouse IgG (B900620; Proteintech) on a rotating wheel overnight at 4°C and then incubated with protein A agarose beads (Beyotime) at 4°C for 10 h. Beads were collected by centrifugation, washed, boiled in 2 × PAGE loading buffers (Beyotime) and analysed by western blotting. The experiments were repeated three times.
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