The following chemicals and materials were used as received: 1,4-benzenedimethanethiol (BDT) (Adamas Reagent Co., Ltd., China), 2,2-dimethoxypropane (DMP) and Dex (Shanghai Macklin Biochemical Co., Ltd., China), IR 780 (Alfa Aesar), fluorescein (Innochem), 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich), Cell Counting Kit-8 (CCK-8) (Topscience Co., Ltd., China); p-toluenesulfonic acid (PTSA) and Nile red (TCI Development Co., Ltd., China); polyvinyl alcohol (PVA) 1788 (alcoholysis degree = 87–89%), bovine serum albumin (BSA) and 1-diphenyl-2-picryl hydrazyl radical (DPPH) (Aladdin Chemistry Co., Ltd., China); dichloromethane (CH2Cl2), toluene, acetone, ethyl acetate, toluene, n-hexane, ethanol and dimethyl sulfoxide (DMSO) (Sinopharm Chemical Reagent Company, China); lactate dehydrogenase (LDH) cytotoxicity kit, ROS assay kit, bicinchoninic acid (BCA) protein assay kit, lipid peroxidation malondialdehyde (MDA) assay kit and dihydroethidium (DHE) (Beyotime Biotechnology, China); enzyme-linked immunosorbent assay (ELISA) kit for interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) (Invitrogen, USA), interleukin-1β (IL-1β) and MPO (Multisciences Biotech, China); Tunel cell apoptosis detection kit and anti-MPO antibody (Servicebio, China). Millipore Milli-Q water with a resistivity of 18.2 MΩ cm−1 was used.
Cell counting kit 8 cck 8
Manufactured by Topscience
Sourced in China
The Cell Counting Kit-8 (CCK-8) is a colorimetric assay used to determine the number of viable cells in cell proliferation and cytotoxicity assays. The kit utilizes a highly water-soluble tetrazolium salt that is reduced by cellular dehydrogenases to an orange-colored formazan dye, which can be measured spectrophotometrically. The amount of the formazan dye is directly proportional to the number of living cells.
Automatically generated - may contain errors
Lab products found in correlation
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Anti mpo antibody, by Wuhan Servicebio Technology (1 mentions)
Tunel cell apoptosis detection kit, by Wuhan Servicebio Technology (1 mentions)
Ir780, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Merck Group (1 mentions)
Milli q water, by Merck Group (1 mentions)
Ros assay kit, by Beyotime (1 mentions)
Bicinchoninic acid bca protein assay kit, by Beyotime (1 mentions)
Fer 1, by Topscience (1 mentions)
Multimode plate reader, by PerkinElmer (1 mentions)
Anti brn3a, by Abcam (1 mentions)
Anti p mlkl, by Abcam (1 mentions)
Bca protein assay kit, by Bioss Antibodies (1 mentions)
Goat anti rabbit igg h l, by ZenBio (1 mentions)
Anti mlkl, by Abcam (1 mentions)
Anti gpx4, by Abcam (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Beyotime (1 mentions)
Jc 1 mitochondrial membrane potential assay kit, by Beyotime (1 mentions)
Goat anti rabbit igg h l alexa fluor 488, by Abcam (1 mentions)
Dulbecco s modified eagle s medium dmem, by Procell (1 mentions)
14 protocols using cell counting kit 8 cck 8
1
Comprehensive Material Characterization Protocol
2
Ferroptosis Induction and Inhibition Assay
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After inoculating cells (5,000 per well) into the 96-well plates, the ferroptosis inducer RSL3 (2.5 µM for H1299, 8 µM for A549, T3646, Top science) or co-treatment with the ferroptosis inhibitor ferrostatin-1 (Fer-1, 2 µM, T6500, Top science) was added 6 hours, then cell viability was measured using a Cell Counting Kit-8 (CCK-8) (C0005, Top Science). Plates were incubated to 60-min CCK8, followed by reading at 450 nm. All experiments were repeated at least three times.
3
Cell Viability Quantification via CCK-8 Assay
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Cell Counting Kit-8 (CCK-8) (Topscience, Shanghai, China) was applied to measure cell viability. Exponentially growing cells were seeded into 96-well plates at a concentration of 5000 cells per well. 36 hours after seeding, CCK-8 solution was added and incubated at 37°C for four hours. Then at 450 nm, the optical density (OD) value was measured.
4
Cell Viability Assay with CCK-8
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Cell Counting Kit-8 (CCK-8) (Topscience, Shanghai, China) was used to measure cell viability. We seeded 5000 cells per well in 96-well plates and treated them with different concentrations of fatostatin and other reagents for 24 h. According to the protocol provided by the supplier, we added 10 μl per well of CCK-8 regents and incubated it for 1 h at 37 °C. One hour later, a Multimode Plate Reader (PerkinElmer, Germany) was used to detect the absorbance value at 450 nm.
5
FFA and Metformin Impact on HaCaT Cells
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HaCaT cells were treated with FFA and metformin. Before the measurement, cells were seeded into 96‐well plates and incubated for 24 hours, and then 10 μL of Cell Counting Kit‐8 (CCK8) (Topscience, Shanghai, China) solution was added and incubated for 1 hour. Absorbance (450 nm) was measured using a quantitative automatic microplate reader.
6
Cell Proliferation Assay with CCK8
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HaCat cells were seeded into 96-well plates at 5 × 10 3 cells/well and incubated for increasing durations (0, 12, 24, 48, and 72 hours) before adding 10 µL of Cell Counting Kit-8 (CCK8) (Topscience, Shanghai, China) solution per well for 1 hour. Absorbance (450 nm) was measured using a quantitative automatic microplate reader.
7
Cell Viability Quantification via CCK-8
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Cell viability was measured using a Cell Counting Kit-8 (CCK-8, Topscience) according to the manufacturer’s instructions. Briefly, cells were seeded in a 96-well plate at a density of 5000 cells per well. The next day, after the treatment of different compounds for a certain time, each well was added with 10 μl of CCK-8 and cultured for 2 hours (37°C, 5% CO2), and the absorbance was detected by using a microplate reader (BioTek Epoch) at 450 nm.
8
Cell Viability Assay using CCK-8 Kit
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For cell viability assay, the Cell Counting Kit-8 (CCK-8) (Topscience, Shanghai, China) was prepared to assess cell viability following the manufacturer’s instructions. One thousand cells/well were plated in 96-well plates and incubated at 37 °C. Cells were exposed to the drugs for 24 h continuously, after which cell viability was assayed by microplate reader (Thermo Fisher Scientific, MA, USA) at 450 nm absorbance. Cell viability was calculated as:
The black group contained only the medium and cells in the control group were cultured with DMSO. All cell-based assays were completed at least in triplicate.
The black group contained only the medium and cells in the control group were cultured with DMSO. All cell-based assays were completed at least in triplicate.
9
Cell Viability, Apoptosis, and Mitochondrial Assays
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Dulbecco's modified Eagle's medium (DMEM) was purchased from Procell (Wuhan, China). Fetal bovine serum (FCS500) was purchased from ExCell Bio (Shanghai, China). Cell counting kit-8 (CCK-8) and Ripa-56 (T7795, 100.00%) were purchased from Topscience (Shanghai, China). Annexin V‐FITC/PI apoptosis detection kit, JC-1 mitochondrial membrane potential assay kit and Hoechst/PI staining buffer were from Beyotime (Shanghai, China). BCA protein assay kit (C05-02001) was purchased from Bioss (Beijing, China). β-actin antibody (66009-1-Ig, 1:20,000) were from Sigma-Aldrich (St. Louis, MO, USA). Anti-Caspase 3 (ab184787), Anti-Bax (ab32503), Anti-Bcl-2 (ab196495), Anti-MLKL (ab243142), p- Anti-MLKL (ab196436), Anti-Brn3a (ab245230, 1:100), Anti-Gpx-4 (ab125066) and Goat Anti-Rabbit IgG H&L (Alexa Fluor 488, ab150077) were obtained from Abcam (Cambridge, UK). Anti-RIP1(#3493S) was purchased from Cell Signaling Technology (CST, Massachusetts, America). Anti-SLC7A11 Polyclonal Antibody (PA1-16893) was from Thermofisher. Anti-IL-6 (EM1701-45, 1:1000) was purchased from HUABIO (Hangzhou, China). Anti RBPMS was from Proteintech (Chicago, America). Goat Anti-Mouse IgG H&L (550017) and Goat Anti-Rabbit IgG H&L (550018) were from Zenbio (China, Chengdu).
10
Comparative Analysis of HA-based Dermal Fillers
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The two HA products were ART FILLER Universal (Fillmed, Paris, Franch) and Restylane II (Q-Med AB, Uppsala, Sweden). The primary antibodies used for IHC were anti-TGF-β (ab215715, Abcam), anti-MMP9 (bs-0397R, Bioss), anti-CD31 (ab28364, Abcam) and used for Western blot analysis were anti-CD44 (ab6124, Abcam), anti-p62 (ab109012, Abcam), anti-LC3 (GTX127375, GeneTex), and anti-Tubulin (BS1699, Bioworld). Senescence β-Galactosidase staining kit was from Beytime Biotechnology (C0602, China). Senescence β-Galactosidase activity kit was from BioLab (SK170-2, China). The Cell Counting Kit-8 (CCK-8) was purchased from TOPSCIENCE (C0005, China), and the Cell-Light EdU Apollo 643 in vitro Kit was form RiboBio (C10310, China). RFP-GFP-LC3 adeno virus was from Hanbio (Shanghai, China).
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