HT-29 and HCT-15 human colon carcinoma cells and MC-38 murine colon carcinoma cells were purchased from China Infrastructure of Cell Line Resources (China) and then cultured in DMEM and RPMI-160 medium, respectively, supplemented with 10% (v/v) fetal bovine serum (FBS) and 100 U/ml streptomycin/penicillin at 37 °C and 5% CO2. Cell Counting Kit-8 (CCK-8) reagent was purchased from Dojindo (Japan). Hoechst 33342 reagent was purchased from Beyotime (China). An Annexin-V FITC apoptosis detection kit (#556547) and FITC BrdU cycle detection kit Part A (#559619) were purchased from BD Biosciences PharMingen (USA). Taxifolin was purchased from Aladdin (China). Antibodies against Bcl-2, Bax, caspase 9/p35/p10, poly (ADP-ribose) polymerase-1 (PARP1), cyclin D1, and beta-actin were purchased from Proteintech. Antibodies against cyclin A2, cyclin B1, p-Akt (S473), p-Akt (T308), pan-Akt, p-ERK (T202/Y204) and pan-ERK were purchased from CST. Antibody against CDK2 was purchased from Abcam. CR powder was provided by the Chinese Medicine Pharmacy of Tianjin Medical University Cancer Institute and Hospital. The concentration of the prepared CR solution was 0.05 g/ml. In addition, for the subsequent relevant biological experiments, the 0.05 g/ml solution was subjected to 0.22 μm filter membrane filtration and sterilization and dilution to the necessary concentrations.
Cell counting kit 8 cck 8 reagent
Manufactured by Dojindo Laboratories
Sourced in Japan, China, United States
The Cell Counting Kit-8 (CCK-8) reagent is a colorimetric assay used for the determination of cell viability and proliferation. It utilizes a water-soluble tetrazolium salt that is reduced by cellular dehydrogenases, producing a yellow-colored formazan dye. The amount of formazan dye generated is directly proportional to the number of living cells, which can be measured using a spectrophotometer.
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Lab products found in correlation
Microplate reader, by Bio-Rad (9 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Microplate reader, by Agilent Technologies (6 mentions)
Microplate reader, by Molecular Devices (3 mentions)
Microplate reader, by Thermo Fisher Scientific (3 mentions)
96 well plate, by Corning (3 mentions)
Hoechst 33342 reagent, by Beyotime (2 mentions)
β actin, by Proteintech (2 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (2 mentions)
Dimethyl sulfoxide (dmso), by Solarbio (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Imark microplate reader, by Bio-Rad (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
Erk1 2, by Cell Signaling Technology (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Multiskan go, by Thermo Fisher Scientific (2 mentions)
160 protocols using cell counting kit 8 cck 8 reagent
1
In Vitro Evaluation of Taxifolin Against Colon Cancer
2
Apoptosis Assay of MAH-infected BMDCs
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For Annexin V and PI staining, BMDCs were infected with MAH (MOI of 1) in the presence or absence of LPS (100 ng/ml) at 37°C. After 24 h, the cells were harvested, washed twice with PBS, and stained using Annexin V and PI apoptosis Detection kit (BD Bioscience, San Jose, CA, USA) according to the manufacturer’s instructions. The cells were analyzed using a LSRII flow cytometer (Becton Dickinson, San Jose, CA, USA). Under the same culture conditions for Annexin V and PI staining, Cell Counting Kit-8 (CCK-8) reagent (Dojindo Laboratories, Tabaru, Japan) was added according to the manufacturer’s instructions to each well for 1 h at 37°C. Viable cells were analyzed by the absorbance at 450 nm, using a microplate ELISA reader.
3
Cell Viability and Apoptosis Assay
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For detection of cell viability, Cell Counting Kit-8 (CCK-8) was applied to this experiment [17 (link)]. Experimental cell suspension was seeded into a 96-well plate. The density was 5 × 103 cells/well. After being fostered for 24 hours, 10 μl Cell Counting Kit-8 (CCK-8) reagent (Dojindo, Kumamoto, Japan) was added to the cell suspension. The absorbance at 450 nm was obtained using a microplate reader (Molecular devices, CA, USA).
flow cytometry was used to analyze the cell apoptosis. Experimental cells were gathered and washed by PBS. About 5 × 105 cells were suspended by 500 μl binding buffer. Then, 5 μl Annexin V-EGFP and 5 μl Propidium Iodide were mixed with cell suspension. Subsequently, cells were stained for 30 minutes in dark. A flow cytometry (Beckman, Fullerton, CA) was utilized to assess cells. The reagents utilized in this assay were from Annexin V-EGFP Apoptosis Detection Kit (Keygen Bio, Nanjing, China) [18 (link)].
flow cytometry was used to analyze the cell apoptosis. Experimental cells were gathered and washed by PBS. About 5 × 105 cells were suspended by 500 μl binding buffer. Then, 5 μl Annexin V-EGFP and 5 μl Propidium Iodide were mixed with cell suspension. Subsequently, cells were stained for 30 minutes in dark. A flow cytometry (Beckman, Fullerton, CA) was utilized to assess cells. The reagents utilized in this assay were from Annexin V-EGFP Apoptosis Detection Kit (Keygen Bio, Nanjing, China) [18 (link)].
4
Evaluating miR-29b Mediated Cell Proliferation
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LX‐2 cells were re‐seeded at a density of 7 × 103 cells per well in 96‐well plates 24 h prior to transfection and divided into three groups: blank group, NC group and miR‐29b transfection group. Cells from each group were seeded in triplicate. Medium was refreshed 24 h after transfection with 10 µl/well Cell Counting Kit‐8 (CCK‐8) reagent (Dojindo Laboratories, Kumamoto, Japan). Cells were incubated in 10% CCK‐8 diluted in normal culture media at 37 °C until visual colour conversion appeared. Proliferation rates were determined at 24, 48 and 72 h post‐transfection, and quantification was performed on a microplate reader set according to the manufacturer's protocol. The proliferation inhibition rate was calculated from the following model: proliferation inhibition rate = [1 − (A450 nmExperimental group − A450nmblank group) / (A450 nmControl group − A450 nmblank group)] × 100%.
5
Cytotoxicity and Apoptosis Assays
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RPMI 1640 medium, fetal bovine serum (FBS), and trypsin-EDTA were purchased from Gibco, USA. Minimum Eagle’s medium (MEM) was obtained from HyClone, USA. Antibiotic/antimycotic solution, kanamycin, puromycin, and dimethyl sulfoxide (DMSO) were obtained from SolarBio, China. DSF was purchased from Selleck, China, whereas CuCl2 was obtained from J&K, China. Phenylmethanesulfonyl fluoride (PMSF), radioimmunoprecipitation assay (RIPA) buffer, and bicinchoninic acid (BCA) reagent were obtained from Beyotime, China. Cell counting kit-8 (CCK-8) reagent was purchased from Dojindo, Japan, whereas AnnexinV-FITC (AV-FITC), propidium iodide (PI) and PI kit were purchased from Roche, Germany. Electrochemiluminescence (ECL) was obtained from Biosharp, China. Lipofectamine 2000 (Lip2000) was purchased from Invitrogen, USA, whereas ROS assay kit was obtained from NJJCBIO, China. Seahorse XF Glycolysis Stress Test and XF Cell Mito Stress Test kits were purchased from Agilent, USA. DeadEnd™ Fluorometric TUNEL System was obtained from Promega, USA, whereas antifade mounting medium with DAPI was purchased from SolarBio, China.
6
Spleen Lymphocyte Proliferation Assay
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Splenic lymphocytes were removed from five mice in each group after euthanasia at two weeks after final immunization. The spleen was pushed through a nylon sieve to harvest the spleen cells. To remove the red blood cells, red blood cell lysis buffer (Sigma, St. Louis, MO, USA) was used. Moreover, DMEM, which was supplemented with 10% fetal bovine serum, was used to resuspend the purified spleen cells. The purified spleen cells were cultured in 96-well plates (2 × 105 cells/well) in triplicate in the presence of 10 μg/mL rTG_200 protein. The negative control comprised cells incubated in complete medium, and the positive control comprised cells incubated in 5 μg/mL concanavalin A (ConA). The plates were incubated for four days at 37°C under 5% CO2. The proliferative activity of spleen lymphocytes was detected using the Cell Counting Kit-8 (CCK-8) reagent (Dojindo, Kumamoto, Japan) following the manufacturer’s protocol. The absorbance was read and recorded at 570 nm. Then, the stimulation index (SI) was calculated using the following formula:
7
Cell Proliferation Assay in U2OS and MG63 Cells
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U2OS and MG63 cells were first seeded into 96‐well tissue culture plates (3 × 103 cells per well). The detection was performed at 0, 24, 48 and 72 hours. After adding 10 µL of Cell Counting Kit‐8 (CCK8) reagent (Dojindo Molecular Technologies, Inc, Kumamoto, Japan) per well and incubating for 2 hours, the absorbance at 450 nm was measured using a Multiscan FC plate reader and analysed with SkanIt for Multiscan FC software (Thermo Scientific).
8
Cell Viability Assay Using CCK-8
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SACC-83 cells were seeded in 96-well plates at 2×103 cells/well in 100 µl of culture medium. After 24 h of incubation, the cells were treated in the presence or absence of the different reagents at the concentrations and for the periods indicated. The medium was then removed and the cells were washed twice with fresh medium. Next, 100 µl of fresh serum-free RPMI 1640 medium containing 10 µl of Cell Counting Kit-8 (CCK-8) reagent (Dojindo, Kumamoto, Japan) was added to each well. After 2 h of incubation at 37°C, the optical density (OD) was recorded on an ELx808 Absorbance Microplate Reader (BioTek, Winooski, VT) at 450 nm. Untreated cells were defined as the negative control, and wells containing the CCK-8 reagent and no cells were used as the blank control. The percentage viability of the control was calculated as (OD of treated cells – OD of blank control)/(OD of negative control – OD of blank control)×100.
9
Nociceptin-Induced Cell Viability Assay
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Three thousand A549 cells were seeded in each well of a 96 well microplate with 100 μL cell culture media. Nociceptin (Tocris, Minneapolis, MN, cat No. 0910) with indicated concentration with or without UFP 101 (R &D, Minneapolis, MN, cat No. 1552) or Ly294002 (MedChem Express, Monmouth, NJ, cat No. HY-10108) were added into the well and co-incubated for 24, 48, and 72 h in a CO2 incubator. Ten microliter Cell Counting Kit-8 (CCK-8) reagent (Dojindo, Kumamoto, Japan) was added into each well and further incubated for 1–2 h. The absorbance was measured at 450 nm with a microplate reader. Each experiment was conducted 3 times.
10
Evaluating Synergistic Effects of Drug Combinations
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PC-3 cells were seeded on 96-well plates (2,000 cells/well in 100 µL), with three replicates used for each concentration. The cells were treated with various concentrations of sorafenib or clofoctol before incubation with 10 µL per well of Cell Counting Kit-8 (CCK-8) reagent (Dojindo Molecular Technologies Inc., Shanghai, China) or Alamar blue (Sigma-Aldrich, MO, USA) solution for 3 hours. Cell viability was estimated by CCK-8 assay or Alamar blue assay.21 (link) The OD was measured using a microplate reader (Thermo Fisher Scientific). The IC50 values were calculated using the GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). The combined effect can be compared to the expected additive effect given by the common formula Eab = Ea + (1-Ea) Eb, where Eab represents the theoretical effect of the two drug combinations and Ea and Eb represent the two drugs.22 (link) To determine the potential mechanism of drug–drug interactions, we used the CompuSyn software (CompuSyn, Cambridge, UK) to calculate the combination index (CI). A CI of <1.0 was considered to be indicative of synergism.
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