IGF-1, BCA Kit, penicillin/streptomycin mix, and Puromycin were purchased from Beijing Soleibao Company (Beijing, China). Akt specific inhibitor (MK-2206) was from MedChem Express (Monmouth, NJ, USA). Adenovirus encoding the human Akt S473D mutant carboxyterminally tagged 3 copies of flag epitope (1.2 × 1010 pfu/mL) and the GFP adenovirus (1.5 × 1010 pfu/mL) were from WZ Biosciences Inc. (Jinan, Shandong, China). Lipofectamine 3000 was from Thermo Fisher Scientific (Waltham, MA, USA). G418 was from Gibco (Grand Island, New York, NY, USA). Transwell chamber and matrigel were from BD Biosciences (San Jose, CA, USA). Protein A/G-agarose was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). CCK8 was from AbMole (Houston, TX, USA). Myc-agarose was Sigma-Aldrich (St. Louis, MI, USA). HyClone Dulbecco’s Modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Cytiva (Marlborough, MA, USA). All antibodies with catalog number and manufactures are listed in Table 1 .
Cck 8
Manufactured by AbMole
Sourced in United States, China
CCK-8 is a colorimetric cell viability assay kit. It is used to measure the number of viable cells in proliferation, cytotoxicity, or cell growth inhibition experiments.
Automatically generated - may contain errors
Lab products found in correlation
Flexstation 3, by Molecular Devices (2 mentions)
Microplate reader, by Bio-Rad (2 mentions)
Ensight, by PerkinElmer (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Mk2206, by MedChemExpress (1 mentions)
Protein a g agarose, by Santa Cruz Biotechnology (1 mentions)
Matrigel, by BD (1 mentions)
Fetal bovine serum (fbs), by Cytiva (1 mentions)
Transwell chamber, by BD (1 mentions)
Transfection reagent, by Thermo Fisher Scientific (1 mentions)
Thermo scientific microplate reader, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Rayto (1 mentions)
Spss 20, by IBM (1 mentions)
Microtiter plate reader, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Cisplatin, by AbMole (1 mentions)
Cck 8, by GraphPad (1 mentions)
Prism 9, by GraphPad (1 mentions)
19 protocols using cck 8
1
Akt Pathway Activation and Inhibition Assays
2
Transfection and Proliferation Assay of gEEC
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The chi-let-7b-5p mimic and inhibitor, as well as the corresponding NC mimic and inhibitor, were bought from the Guangzhou Ruibo biological company. Immortalized gEECs were obtained as previously described [61 (link)] The cells were transfected once they had fused to 50%. In brief, chi-let-7b-5p mimic was diluted with serum-free medium Opti, mixed with transfection reagent (ThermoFisher Scientific, Waltham, MA, USA), incubated at room temperature for 20 min, added to the appropriate well drop by drop, and collected after 48 h for follow-up operation. Following transfection, 10 μL CCK8 (AbMole Bioscience Inc., Houston, TX, USA) was added to each well at the appropriate time in order to study the impact of chi-let-7b-5p on the proliferation of gEEC. Incubation was then continued for another two hours. The absorbance of gEEC at 450 nm was measured using a Thermo Scientific Microplate Reader (ThermoFisher Scientific, Waltham, MA, USA).
3
Evaluating IDA and Olaparib on Cell Viability
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K562 and THP1 cells were seeded into 96-well plates at a density of 1×10
4 cells/well. Cells were cultured with different concentrations of IDA in the absence or presence of Olaparib (2 μM) for 24 h. Subsequently, cell viability was measured using a cell counting kit-8 (CCK-8; AbMole, Shanghai, China). Briefly, K562 and THP1 cells were incubated with 10 μL CCK-8 reagent and then further incubated at 37°C in 5% CO
2 for 2 h. Optical density was measured at 450 nm using a microplate reader (Rayto, Shenzhen, China).
4 cells/well. Cells were cultured with different concentrations of IDA in the absence or presence of Olaparib (2 μM) for 24 h. Subsequently, cell viability was measured using a cell counting kit-8 (CCK-8; AbMole, Shanghai, China). Briefly, K562 and THP1 cells were incubated with 10 μL CCK-8 reagent and then further incubated at 37°C in 5% CO
2 for 2 h. Optical density was measured at 450 nm using a microplate reader (Rayto, Shenzhen, China).
4
Cytotoxicity Assay of DCL in RAW264.7 Cells
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RAW264.7 cells (1.5 × 104 cells/100 μl) seeded in 96-well plates were treated with various concentrations of DCL for 2 h, and then stimulated with LPS (0.5 μg/ml) and IFNγ (10 ng/ml) for 24 h. Next, 10 μl of Cell Counting Kit-8 (CCK-8) (AbMole, Shanghai, China) solution was added to each well for 1 h at 37°C. The optical density (OD) at 450 nm was analyzed using a multifunctional microplate reader (FlexStation 3; Molecular Devices).
5
Cell Viability Assay for Lymphoma Cells
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1 × 104 SU-DHL-2 or SU-DHL-6 cells were incubated into the 96‐well plates in 100 μL serum medium per well. A series of two-fold dilutions of decoctions were carried out to give concentrations of 10, 5, 2.5, 1.25, 0.625 and 0.313 mg/ml. Untreated tumor cells and complete medium (blank control) were left as control groups. After 48 h incubation, 10 µL of CCK8 (AbMole Bioscience) was added into all wells and the plates were further incubated for 2 h. Then, the plates were read at 450 and 630 nm as reference wavelength. After background subtraction, half maximal inhibitory concentration (IC50) was calculated using the SPSS 20.0 (IBM, United States). The experiments were repeated in triplicate for each inhibitor concentration.
6
Cell Viability Assay with CCK8
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Cell viability was assessed by using the cell counting kit 8 (CCK8; M4839, Abmole) according to the manufacturer’s instructions. Cells were seeded in 96-well plates (20,000 cells per well) and treated with 10 μl erastin or PBS in the incubator for 24 h. Then the culture medium was replaced with 100 μl fresh supplemented DMEM containing 10 μl CCK8 in each well of the plate. The cells were incubated in the cell incubator for 2 h and then the absorbance at 450 nm was measured using the microplate reader.
7
Cell Proliferation Assay in Osteosarcoma
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Osteosarcoma cells (5000 cells/well) were seeded in 96-well plates and detected for cell proliferation using Cell Counting Kit-8 (CCK-8) assay according to the manufacturer's instructions. Briefly, the cells were incubated with 10 µL of CCK-8 (AbMole BioScience, Houston, TX, USA) at 37℃ for 4 h. Absorbance at 450 nm was evaluated using a microtiter plate reader (Thermo Fisher Scientific). Each group was tested with three replicates.
8
Evaluating Bladder Cancer Cell Viability
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T24 and 5637 cells were seeded in 96-well plates with RPMI-1640 medium containing 10% FBS, followed by treatment with BAC and GEM. The viability of BC cells was evaluated by the Cell Counting Kit-8 (CCK-8, Abmole, Houston, USA) assay according to the manufacturer’s instructions. Cells were treated with BAC for 72 h alone or followed by GEM for 48 h, and 10 μL of CCK-8 solution was then added to each well and incubated with the cells for 0.5–1.5 h at room temperature. Then, the absorbance at 450 nm was determined by a microplate reader (Bio-Tek, Winooski, USA). The final data were normalized to the optical density (OD) at 450 nm value of untreated cells. Cell viability (%) was calculated as follows: .
For the monoclonal cell colony forming assay, T24 and 5637 cells were cultured in RPMI-1640 medium with 10% FBS at 37 °C after treatment with BAC for 72 h alone or followed by GEM for 48 h, then 500 treated and untreated BC cells were seeded in 12-well plates. Untreated cells were used as the control group. After 14 days of incubation, the colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet for 30 min. The colony forming efficiency was quantified by ImageProPlus 6.0 software (Media Cybernetics, Rockville, USA).
For the monoclonal cell colony forming assay, T24 and 5637 cells were cultured in RPMI-1640 medium with 10% FBS at 37 °C after treatment with BAC for 72 h alone or followed by GEM for 48 h, then 500 treated and untreated BC cells were seeded in 12-well plates. Untreated cells were used as the control group. After 14 days of incubation, the colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet for 30 min. The colony forming efficiency was quantified by ImageProPlus 6.0 software (Media Cybernetics, Rockville, USA).
9
Evaluating FFA and ATL III Cytotoxicity
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The cells (5×103 cells/well) were seeded in 96-well plates and cultivated overnight, and then cells were incubated with different concentrations of FFAs (0-1.5 mM) or ATL III (0-200 μg/ml) for 24 h or 48 h. Next, the cell counting kit-8 (CCK-8, AbMole Bioscience, USA) solution was added into the culture medium (10 μl/well), and the cultures were incubated for 2 h at 37 °C. The absorbance was measured at 450 nm by Microplate Reader (Bio-Rad; Hercules, CA, USA).
10
Glucose Cytotoxicity Evaluation in BPTCs
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The BPTCs were inoculated in 96-well plates; after this, the cells would grow adductively and then treated with 200 µL medium (Pronox, Wuhan, China) containing different concentrations 1, 2, 4, 8, and 16mg/mL of glucose (D-glucose, ≥99.5%, St. Louis, MO, USA) for 24 h, then observed cell morphology by an inverted microscope. 100 µL of the medium was retained and 10 µL of CCK-8 (Abmole, Shanghai, China) was added to each well. The values of optical density (OD) 450 nm were detected by a microplate reader (Bio-Rad, Hercules, CA, USA) after incubation for 1 h at 37 °C in a 5% CO2 cell incubator.
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