The 3T3-L1 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Dulbecco’s modified Eagle’s medium (DMEM), glutamine, penicillin-streptomycin, fetal bovine serum (FBS), bovine calf serum (BCS), and TRIzol reagent were obtained from Invitrogen (Carlsbad, CA, USA). The cell count kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). An assay kit for TG was obtained from Asan Pharmaceutical Co. (Seoul, Korea). The GPDH activity assay kit was procured from Takara (Kyoto, Japan). The bicinchoninic acid (BCA) protein assay kit was obtained from Thermo Scientific (Pittsburgh, PA, USA). The Puregene DNA isolation kit was purchased from Qiagen (Chatsworth, CA, USA). M-MLV reverse transcriptase and AccuPower® 2X GreenStarTM qPCR Master Mix were purchased from Bioneer (Daejeon, Korea). The AMPK Kinase Assay kit was purchased from Cyclex (Nagano, Japan) and Isorhamnetin was obtained from Sigma Co. (St Louis, MO, USA)
Cell count kit 8 cck 8
Manufactured by Dojindo Laboratories
Sourced in Japan
The Cell Count Kit-8 (CCK-8) is a colorimetric assay for the determination of cell viability and proliferation. It utilizes a tetrazolium salt that is reduced by living cells, resulting in the formation of a water-soluble formazan dye that can be measured spectrophotometrically.
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Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Gpdh activity assay kit, by Takara Bio (2 mentions)
Glutamine, by Thermo Fisher Scientific (2 mentions)
Bicinchoninic acid bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
3t3 l1 cells, by American Type Culture Collection (2 mentions)
Assay kit for tg, by Asan Pharmaceutical (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Accupower 2x greenstartm qpcr master mix, by Bioneer (1 mentions)
M mlv reverse transcriptase, by Bioneer (1 mentions)
Puregene dna isolation kit, by Qiagen (1 mentions)
Isorhamnetin, by Merck Group (1 mentions)
Sp8 confocal microscope, by Leica camera (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Trypsin edta solution, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Keygen Biotech (1 mentions)
15 protocols using cell count kit 8 cck 8
1
Adipogenesis Assay with 3T3-L1 Cells
2
Viability of NSCs on Nanostructured rGO Microfibers
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To assess the viability of the NSCs on nanostructured rGO microfibers, NSCs were cultured on nanostructured rGO microfibers for 3 days under proliferation conditions, and the viability state of the cells was checked by staining the cells with the LIVE/DEAD Cell Imaging kit for mammalian cells (Life Technologies, USA) according to the manufacturer’s instructions.
A Cell Count Kit-8 (CCK-8, Dojindo Molecular Technology) was used to quantitatively evaluate cell viability on tissue culture plates, graphene films and nanostructured rGO microfibers after cultivation for 1, 3 and 5 days.
To further demonstrate the cytocompatibility of the nanostructured rGO microfibers, after the NSCs were cultured for 5 days under proliferation condition, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 1% bovine serum albumin (BSA, Sigma, USA) solution at room temperature. To check the stemness of the NSCs and the location and distribution of the cells, the fixed cells were incubated with Alexa Fluor 488 conjugated Nestin (1:100, Millipore, USA) for 60 min and the nuclei were stained by 4′ 6-diamidino-2-phenylindole (DAPI, 300 nM, Life Technologies, USA) for 10 min. Finally, the cells were washed with TPBS three times before being examined with a Leica Confocal Microscope SP8.
A Cell Count Kit-8 (CCK-8, Dojindo Molecular Technology) was used to quantitatively evaluate cell viability on tissue culture plates, graphene films and nanostructured rGO microfibers after cultivation for 1, 3 and 5 days.
To further demonstrate the cytocompatibility of the nanostructured rGO microfibers, after the NSCs were cultured for 5 days under proliferation condition, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 1% bovine serum albumin (BSA, Sigma, USA) solution at room temperature. To check the stemness of the NSCs and the location and distribution of the cells, the fixed cells were incubated with Alexa Fluor 488 conjugated Nestin (1:100, Millipore, USA) for 60 min and the nuclei were stained by 4′ 6-diamidino-2-phenylindole (DAPI, 300 nM, Life Technologies, USA) for 10 min. Finally, the cells were washed with TPBS three times before being examined with a Leica Confocal Microscope SP8.
3
Synthesis and Characterization of Gold Nanoparticles
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HAuCl4·4H2O (48%), AgNO3 (99%), hydroquinone (98%), and NaBH4 (99%) were obtained from Sinopharm Chemicals Reagent Co. Ltd. CTAB (99%) and sodium dodecyl sulfate (SDS, 99%) were purchased from Sigma-Aldrich. The Dulbecco's modified Eagle's medium with high glucose (H-DMEM), fetal bovine serum (FBS), penicillin–streptomycin solution and trypsin–EDTA solution were obtained from Gibco. The Cell Count Kit-8 (CCK-8) used for cell viability was purchased from Dojindo Laboratories. CD47 antibody was purchased from Proteintech. The Annexin V-FITC/PI kit used for flow cytometry was purchased from Sungene Biotech Co. Propidium iodide (PI) used for staining cells was the product of Key Gen BioTech. All the other chemicals were analytical grade and used as received.
4
Cell Proliferation Assay with CCK-8
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Cell Count Kit-8 (CCK-8) (Dojindo Molecular Technologies, Tokyo, Japan) was used to evaluate cell proliferation. PC3 and DU145 cells stably transfected with pCDH-miR-4638-5p, pCDH-miR-4638-5p sponge, pCDH-shKidins220, pCDH-shAKT and pCDH-vector controls were seeded into 96-well plates at density of 2 × 103 cells per well and cultured for 1 day, 2 days, 3 days, 4 days, 5 days and 6 days. Each well of cells were then cultured in 20 μl of CCK-8 solution in addition to the 200 μl of culture medium for 1.5 hours at 37°C. Absorbance at 450 nm was measured using a Vmax microplate spectrophotometer (Molecular Devices). LNCaP cells transfected with pCDH-miR-4638-5p sponge and pCDH-vector control were seeded into 96-well plates (also 2 × 103 cells per well) in the RPMI 1640 medium supplemented with 10% charcoal-stripped FBS and cultured for 1 day, 2 days, 3 days, 4 days and 5 days. Then the cell proliferation ability was evaluated using CCK-8 solution as described above.
5
Evaluation of L929 Cell Proliferation
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Dulbecco's Modified Eagle's Medium (DMEM, Gibco), 1% penicillin/streptomycin (Gibco), and 10% fetal bovine serum (FBS, Gibco) were used to culture the L-929 fibroblasts in a 25 cm2 flask, and the cells were incubated at 37°C in 5% CO2 atmosphere. Cell Count Kit-8 (CCK-8, Dojindo Molecular Technologies, Inc. Japan) was used to evaluate the proliferation of the cells. Briefly, we cultured L929 cells in a naked 96-well disposable plate coated with parylene-C film on an initial density of 1 × 106 cell/well under the log phase of growth. The cell viability was evaluated every day for 1, 2, and 3 days. Then, 210 μL of complete medium including 10 μL of CCK-8 reagent were added after the culture medium was removed, and the cells were washed by phosphate-buffered saline solution (PBS). After incubation for 1.5 h, 150 μL culture supernatant was transferred into a 96-well plate. A synergy H1 hybrid multimode microplate reader (BioTek Instruments Inc., USA) installed with the Gen5 software (version: 2.04) was employed to measure the absorbance at 450 nm.
6
3T3-L1 Adipocyte Differentiation Protocol
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The 3T3-L1 cells were obtained from American Type Culture Collection (Manassas, VA, USA). Dulbecco’s modified Eagle’s medium (DMEM), glutamine, penicillin-streptomycin, fetal bovine serum (FBS), and TRIzol reagent were obtained from Invitrogen (Carlsbad, CA, USA). The cell count kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). An assay kit for TG was obtained from Asan Pharmaceutical Co. (Seoul, Korea). The GPDH activity assay kit was from Takara (Kyoto, Japan). The bicinchoninic acid (BCA) protein assay kit was obtained from Thermo Scientific (Pittsburgh, PA, USA). Universal SYBR® Green PCR Master Mix was purchased from Qiagen (Chatsworth, CA, USA). M-MLV reverse transcriptase was purchased from Promega (Madison, WI, USA). The NO assay kit was purchased from Thermo Scientific (Pittsburgh, PA, USA).
7
Biomaterial Fabrication and Characterization
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Sodium hyaluronate was purchased from Yuanye Bio-Technology Co. Ltd (Shanghai, China). Ethylene glycol, sodium periodate (100–150 kDa), rhodamine B, and polyvinyl pyrrolidone were purchased from Aladdin (Shanghai, China). 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA), 4′, 6-diamidino-2-phenylindole (DAPI), potassium ferricyanide, and gelatin were obtained from Sigma. Cy5 was bought from Ruixibio (Xi’an, China). Cell Count Kit-8 (CCK-8) and Live/dead kit were acquired from Dojindo Molecular Technologies (Kumamoto, Japan). The JC-1 dye and Annexin V-FITC Apoptosis Detection Kit was bought from Solarbio (Solarbio, China). Antibodies of MMP3 (ab52915) and were purchased from Abcam (Abcam, UK). Collagen II (sc-52658) was purchased from Santa cruz (Santa cruz, USA). Aggrecan (13,880-1-AP), MMP13 (18,165-1-AP) and SOX9 (67,439-1-Ig) was purchased from Proteintech (Proteintech, China).
8
Cell Proliferation Assay Protocol
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MSC proliferation was determined using the Cell Count Kit 8 (CCK-8) assay (Dojindo, Japan) according to the recommendation by the manufacturer. 2.5 × 103 cells/well were seeded into a 96-well plate. The absorbance at 450 nm was determined by the multiplate reader Sunrise (Tecan, Austria). Cell proliferation was represented as a percentage and normalized to the control (DMEM). Three experiments were performed in triplicate.
9
Quantifying MC3T3-E1 Cell Proliferation and Differentiation on Scaffolds
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To quantify MC3T3-E1 cell proliferation on scaffolds, we used the Cell Count Kit-8 (CCK-8, Dojindo, Japan) on days 1, 3, and 7 after seeding. CCK-8 solution was diluted 1 : 10 with culture medium and added to samples, which were then incubated for 3 hr at 37°C. The optical densities of culture supernatants were then measured at 450 nm using a microplate reader (Epoch, BioTek, VT, USA). ALP activity was quantified using p-nitrophenyl phosphate (pNPP; Sigma Aldrich) on days 3, 7, and 14 after seeding. Samples were lysed (RIPA lysis buffer) and incubated in pNPP solution at 37°C for 30 min, and ALP activity was measured at 405 nm using a microplate reader and quantified using a p-nitrophenol standard. To estimate calcium deposition on scaffolds, samples were fixed with 4% paraformaldehyde and stained with 2% alizarin red solution (pH 4.2) for 10 min at room temperature on days 3, 7, and 14 after seeding. The alizarin red was extracted from samples using 10% cetylpyridinium chloride and quantified by measuring absorption at 570 nm using a microplate reader.
10
Anti-inflammatory effects of LPS in HepG2
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HepG2 (JCRB1054) was obtained from the Human Science Research Resources Bank (Osaka, Japan). The Dulbecco's Modified Eagle's Medium (DMEM) was purchased from Wako (Osaka, Japan). The Cell Count Kit-8 (CCK-8) and the Cytotoxicity Lactate dehydrogenase (LDH) Assay Kit-WST were obtained from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). LPS was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against IκB, NF-κB p65, TLR4, MyD88 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Phospho-IκBα Ser-32/36 rabbit polyclonal antibody and phospho-NF-κB p65 Ser-536 (93H1) rabbit monoclonal antibody were purchased from Enzo Life Sciences (Farmingdale, NY, USA) and Cell Signaling (Danvers, MA, USA), respectively. Goat anti-mouse immunoglobulin G (IgG) peroxidase antibody, goat antirabbit IgG peroxidase antibody and goat anti-mouse IgG fluorescein isothiocyanate-conjugated (FITC-conjugated) antibody were obtained from Sigma-Aldrich. Unless otherwise stated, all the other reagents and chemicals were of the highest grade commercially available.
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