An HTS assay based on the cytopathic effect (CPE) was performed as previously reported (Tang et al., 2020a (link)). Briefly, RD cells were infected with EV-A71 at a multiplicity of infection (MOI) of 0.1, followed by the treatment of compounds (10 μM). The antiviral activities of compounds were measured by a CCK8 kit based on cell viability. The antiviral effect of potential compounds was further assessed by western blotting. The cytotoxic effect was tested by a CCK-8 kit (Vazyme Biotech) as previously reported and the selective index (SI) was calculated based on the EC50/CC50values.
Cck 8 kit
Manufactured by Vazyme
Sourced in China
The CCK-8 kit is a cell counting and cytotoxicity assay used to measure cell viability and proliferation. It contains a water-soluble tetrazolium salt that is reduced by living cells to produce a colored formazan dye, which can be quantified using a spectrophotometer.
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Lab products found in correlation
Enspire, by PerkinElmer (6 mentions)
Cell light edu apollo567 in vitro kit, by RiboBio (4 mentions)
Multiskan fc, by Thermo Fisher Scientific (4 mentions)
Microplate reader, by Thermo Fisher Scientific (4 mentions)
Prism 8, by GraphPad (3 mentions)
Lipofectamine 3000 transfection kit, by Thermo Fisher Scientific (3 mentions)
Elx800, by Agilent Technologies (2 mentions)
Glass botttom cell culture dishes, by NEST Biotechnology (2 mentions)
Accuri c6 flow cytometer, by BD (2 mentions)
Microplate reader, by Bio-Rad (2 mentions)
Annexin v 7aad apoptosis assay kit, by BD (1 mentions)
Cba kit, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Microplate reader at 450 nm, by Thermo Fisher Scientific (1 mentions)
Bortezomib, by AbMole (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Mg132, by AbMole (1 mentions)
Depc water, by Sangon (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
59 protocols using cck 8 kit
1
CPE-based HTS Assay for EV-A71 Antivirals
2
Quercetin's Anticancer Potential on MIA PaCa-2 and Huh7 Cells
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To evaluate the antitumor potential of quercetin, the viability of MIA PaCa-2 and Huh7 cells after treatment with quercetin was assessed using the Cell Counting Kit-8 (CCK-8) assay. Cells (2,500 cells per well) were seeded in 96-well plates, and after 24 h they were treated with different concentrations of quercetin. After 72 h of culture, the CCK-8 reagent was added to each well according to the instructions provided by the manufacturer of the CCK-8 Kit (Vazyme Biotech Co., Ltd.). The absorbance was measured at 450 nm using a microplate reader (MULTISKAN FC, Thermo Fisher Scientific). Cells treated with DMSO served as the control. The IC50 values were calculated by GraphPad Prism 8.4.0.
3
Morroniside Modulates C2C12 Myogenesis
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C2C12 cells were treated with different concentrations of morroniside (0–640 μg/ml) for 48 h, then the cell survival was examined by CCK8 kit (Vazyme, catalog A311-02). Primary myogenic cells (CD45−; CD31−; CD11b−; Sca1−) were isolated according to our previously published method (Li et al., 2020 (link)). Isolated myogenic cells (CD45−CD11b−CD31−Sca1−) or C2C12 cells (a gift from Prof. Yiting Zhou, Zhejiang University) were seeded in growth medium (10% FBS, 1% Pen/Strep, 1% Glu in DMEM) until cell confluency reached ∼70%. The cells were induced with differentiation medium (2% horse serum,1%Pen/Strep, 1% Glu in DMEM) with 5, 10, or 50 ng/ml TNFα with or without lower dose of morroniside (Mor-L, 80 μg/ml) or higher dose of morroniside (Mor-H, 160 μg/ml) for 8–72 h, and the differentiation medium with TNFα and/or morroniside was changed every 12–48 h.
4
Cytotoxicity Assay of iCRT14
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The proliferation and cytotoxicity of iCRT14 on CEF and LMH cells were determined using a cell counting kit, CCK-8 kit (Vazyme, Nanjing, China). In brief, 1.0 × 104/well CEF cells were seeded in 96-well plates, and 100 μL of iCRT14 at 0 and 10 μM in DMEM maintenance medium were added to each well. After incubating for 24, 48, 72, and 96 h in a 37 °C incubator, CCK-8 solution (10 μL) was added to each well. Incubation was continued for 1 h, and the absorbance at 450 nm was measured.
5
Characterizing CAR-T Cell Proliferation
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Cell lines and primary cells were assayed for cell proliferation using the CCK8 kit (Vazyme Biotech, Nanjing, China), and cell proliferation inhibition rate (%) = (OD treated − OD blank)/(OD control − OD blank) × 100%. Apoptosis was detected by AnnexinV/7-AAD apoptosis assay kit (BD Biosciences, USA), and the apoptosis rate was detected by flow cytometry after cell staining. The proliferation of T cells was detected by CFSE. CD22-CAR-transduced T cells and mock-transduced T cells were labeled with CFSE separately and co-cultured with different target cells at a 1:1 ratio in 24-well plates with IL-2-free T cell complete medium (TCM). 4 days later, Cells were stained with CD3-APC/Cy7, and the cell division index (CDI) was calculated to compare the proliferation differences of T cells in each group.
6
Proliferation and Edu-labeling of PGCs
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The proliferative capacity was detected using CCK-8 kit (A311-01, Vazyme, Nanjing, China) according to the manufacturer’s instruction. Briefly, 100 μL cell solution (1 × 106/mL) of PGCs at logarithmic growth stage was seeded in 96-well plates in triplicate. Then, 10 μL of CCK-8 solution was added, followed by the incubation at 37 °C and 5% CO2 for 2 h. The absorbance at 450 nm was read using microplate reader (TECAN SPARK, Shanghai, China).
After the Edu-labeling (C10310-1, Ribobio, Guangzhou, China), fixation, and permeabilization, PGCs were incubated with Click reaction solution for 30 min at room temperature in dark and then stained with Hoechst33342 for 10 min at room temperature in dark. Fluorescence was observed under fluorescence microscope (LEICA DMi8, Beijing, China).
After the Edu-labeling (C10310-1, Ribobio, Guangzhou, China), fixation, and permeabilization, PGCs were incubated with Click reaction solution for 30 min at room temperature in dark and then stained with Hoechst33342 for 10 min at room temperature in dark. Fluorescence was observed under fluorescence microscope (LEICA DMi8, Beijing, China).
7
Cell Viability Assessment of Transfected DPCs
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For the CCK-8 assay, we planted DPCs using a 96-well cell culture plate. After the DPCs reached 30% density, cells were transfected. According to the instruction manual of CCK-8 Kit (Vazyme, Nanjing, China), cell viability was detected at 12 h (after DPC transfected), 24 h, 36 h, and 48 h. The multi-mode micropore detection system (EnSpire, PerkinElmer, Waltham, MA, USA) was used to detect the absorbance at 450 nm.
8
Immunomodulatory Cytokine Profiling
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The lymphocyte proliferation was determined every day from the first to the fifth day using a CCK8 kit (Vazyme Biotech, China). The optical absorbance was measured at 450 nm by a microplate reader. The cytokines in the culture medium were analyzed using a CBA kit (BD Bioscience, United States); the assay was carried out according to the manufacturer’s instructions. Data were acquired with a BD FACS Calibur flow cytometer and analyzed with the FCAP array software. IL-2, IL-4, IL-6, IFN-g, TNF-a, IL-17, and IL-10 were recorded and compared.
9
Curcumin Cytotoxicity Evaluation in cBMSCs
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The cellular viability of cBMSCs was determined using the CCK-8 kit (Vazyme Biotech Co., Ltd., Nanjing, China). cBMSCs were pre-cultured in a 96-well plate for 24 h. cBMSCs were treated with Cur at different concentrations (0.1, 0.5, 1, 5, and 10 μmol/L) for 12 h, 24 h, 48 h, and 72 h. Cells were treated with 0.1% of DMSO, which was used as a control. After incubating with 10 μL of CCK-8 solution per well for 2 h, the optical density was measured by a microplate reader at 450 nm (Thermo Scientific, Waltham, MA, USA). The relative cell viability was calculated in accordance with the manufacturer’s instructions.
10
Comprehensive Experimental Protocols for Cell Culture Research
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M199 medium was purchased from Hyclone (SH30253.01). ECM was purchased from Sciencell (1001). Fetal bovine serum was purchased from Gibco (10099141). DMSO was purchased from sigma (D2650). Trizol Reagent was purchased from TAKARA (9108). Protein marker (P0076) and SDS-PAGE (P0690) were purchased from Beyotime. PVDF membrane was purchased from BBI (c62-393–0100). BSA kit (C102301-0002) and DEPC water (B501005-0500) were purchased from Sangon. The Matrigel matrix was purchased from CORNING (356234). CCK-8 kit was purchased from Vazyme (A311-01/02). Flow cytometry kit was purchased from Beyotime (C1062S). Collagenase I was purchased from Maokangbio (MX1001-1000MG). MG132 and bortezomib were purchased from AbMole (M1902 and M1686). 1,6-HD and 2,5-HD were purchased from Sigma-Aldrich (240117 and H11904).
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