To evaluate the acute toxicity of Dpo48 toward human in vitro, HuH-7 (human liver cancer), A549 (human lung carcinoma) and 293T (human embryonic kidney) cell lines were obtained from ATCC (VA, United States) and used in this research. All cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, NY, United States) supplemented with 10% fetal bovine serum (FBS; Gibco) under a humidified atmosphere of 5% CO2 at 37°C, and sub-cultured every 2 days. The cytotoxicity of Dpo48 was determined by MTT assay according to previously methods with minor modifications (Oliveira et al., 2018 (link)). Briefly, cells were added into 96-well microtiter plates (5 × 103 cells/well) and incubated at 37°C for 24 h. The culture medium was removed, and cells were washed with PBS (pH = 7.4). Then, a final concentration of 7.5 μg/mL Dpo48 or PBS was added to cells and incubated at 37°C for 24 h. The toxicity of Dpo48 toward cells was conducted using the MTT Cell Proliferation and Cytotoxicity Assay Kit (Solarbio). Quantification of soluble formazan produced by cellular reduction of MTT was measured at 540 nm using a Synergy HT Multi-Detection Microplate Reader. The experiments were performed in triplicate.
Mtt cell proliferation and cytotoxicity assay kit
Manufactured by Solarbio
Sourced in China
The MTT Cell Proliferation and Cytotoxicity Assay Kit is a colorimetric assay used to measure cell proliferation and cell cytotoxicity. The kit utilizes the tetrazolium dye MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) as the detection reagent.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Fm4 64, by Thermo Fisher Scientific (3 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Reactive oxygen species assay kit, by Beyotime (2 mentions)
Annexin 5 fitc apoptosis detection kit, by Beyotime (1 mentions)
Tnf α enzyme linked immunosorbent assay elisa kits, by Neobioscience (1 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (1 mentions)
Caspase 3 activity assay kit, by Beyotime (1 mentions)
Spss statistics v 16.0, by IBM (1 mentions)
E0228, by Beyotime (1 mentions)
Microplate spectrophotometer, by Bio-Rad (1 mentions)
Dimethyl sulfoxide (dmso), by Biosharp (1 mentions)
Cd4 pe, by BioLegend (1 mentions)
Cd19 pe, by BioLegend (1 mentions)
Cd8 fitc, by BioLegend (1 mentions)
Immunoglobulin g assay kit, by Nanjing Jiancheng (1 mentions)
Immunoglobulin m assay kit, by Nanjing Jiancheng (1 mentions)
Cd3 fitc, by BioLegend (1 mentions)
Spectra max plus absorbance microplate reader, by Molecular Devices (1 mentions)
32 protocols using mtt cell proliferation and cytotoxicity assay kit
1
Evaluating Acute Toxicity of Dpo48 in Human Cell Lines
2
Evaluating Byakangelicin Cytotoxicity and HNE-induced Apoptosis
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The LX‐2 human hepatic stellate cell and HepG2 hepatocyte cell line were seeded in 96‐well plates with DMEM, which included 2% and 10% FBS, respectively, for 24 hours. Then, we exposed the cells to different concentrations of byakangelicin for 24 hours. We used MTT Cell Proliferation and Cytotoxicity Assay Kit (Solarbio Technology) to detect the cytotoxicity. For 4‐hydroxynonenal (HNE) induction, HepG2 cell apoptosis was detected by using Annexin V‐FITC Apoptosis Detection Kit (Beyotime Biotechnology).
3
MTT Cell Proliferation Assay Protocol
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MTT Cell Proliferation and Cytotoxicity Assay kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) was used to detect cell proliferation, according to the manufacturers protocol. The cell suspension was seeded in a 96-well plate at a density of 2×104/ml (100 µl total). Each group was placed in 5 wells. The cells were cultured at 37°C in a 5% CO2 incubator. On days 1, 2, 3, 4 and 5, cells were incubated with 10 µl 5 mg/ml MTT solution for 4 h at 37°C. Next, 100 µl dimethyl sulfoxide was added to each well, and the plates were shaken for 5-10 min. The absorbance at 490 nm was analyzed using a microplate reader (Thermo Fisher Scientific, Inc.) to plot the growth curve.
4
PRRSV Infection and Inhibition Assay
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MARC-145 cells (ATCC CRL-12231) are a PRRSV permissive cell line derived from African green monkey kidney cells, which were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with a 10% fetal bovine serum (FBS, Gibco) at 37°C under a humidified atmosphere of 5% CO2 (50 (link)). The JXA1-like PRRSV strain CH-YY (GenBank Accession No. MK450365) and NADC30-like PRRSV strain CH-WH-2019-1 (GenBank Accession No. MK450333) were used in this study. Tylvalosin tartrate (TVN; ≥ 85%) was purchased from the three major producers A, B, and C in the world. Poria cocos polysaccharides (PCP; ≥ 90%) powder was provided by Wuhan HVSEN Biotechnology Co., Ltd. (Wuhan, China). MTT Cell Proliferation and Cytotoxicity Assay Kit were purchased from Beijing Solarbio Science & Technology (Beijing, China). Caspase 3 Activity Assay Kit was purchased from Beyotime Institute of Biotechnology (Shanghai, China). Transcriptor First Strand cDNA Synthesis Kit and SYBR Green Master (2×) with ROX were purchased from Roche (Mannheim, Germany). Porcine IL-6, IL-8, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits were purchased from NeoBioscience Technology (Shenzhen, China). The primers used in this study are listed in Table 1 .
5
CaM Protein Activation and Cytotoxicity Assay
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The activity of the CaM protein was assayed as the ability of the protein to activate phosphodiesterase (PDE) (Sigma, St. Louis, MO, USA), which hydrolyses cAMP (Sigma) to 5′-AMP [30] (link). Then, 5′-AMP was broken down into adenosine and orthophosphate (Pi) by 5′-nucleotidase (Sigma), which was measured using spectrophotometry at 660 nm. An MTT Cell Proliferation and Cytotoxicity Assay Kit (Solarbio, Shanghai, China) were used to determine the cytotoxicity of the compound following the manufacturer’s protocol [31] (link). The median lethal concentration (LC50) for the assay was analysed with IBM SPSS Statistics (v.16.0).
6
MTT Cell Viability Assay Protocol
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The viability of HGC‐27 or SNU‐1 cells was analyzed using an MTT Cell Proliferation and Cytotoxicity Assay Kit (M1020) from Solarbio (China). HGC‐27 or SNU‐1 cells were cultured routinely for 24, 48, or 72 hours in a 96‐well plate, and then, 90 μl of fresh medium and 10 μl of MTT reagent were added to each well to further culture the cells for 4 hours. Afterward, 110 μl of Formazan solution was added and shaken for 10 minutes. Finally, the absorbance was measured at 490 nm using a microplate absorbance reader (E0228) from Beyotime (China).
7
Evaluating Cell Viability with MTT Assay
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The viability of transfected MKN45 and MKN74 cells with si-CNALPTC1 and pcDNA3.1-CNALPTC1, respectively, was calculated with the aid of the MTT Cell Proliferation and Cytotoxicity Assay Kit (Solarbio, Beijing, China). Trypsin was utilized to detach cells in the logarithmic growth phase, and the cell suspension was prepared after performing transfection. Subsequently, the cells were seeded at 3×105 cells/well onto 96-well plates, with 200 µL medium added in all the wells and marked, and incubation was performed for 24 hours with 5% CO2 at a temperature of 37 ℃. After incubation, the removal of suspension was performed, and the solution was replaced with 200 µL dimethyl sulfoxide (DMSO; BioSharp, Hefei, China) for substrate dissolution for 10 minutes. The absorbance was evaluated using a microplate spectrophotometer (Bio-Rad, Hercules, CA, USA) at 490 nm.
8
Quantifying P. gingivalis Cytotoxicity on HGFs
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HGFs were seeded into 96-well plates at a density of 1 × 104 cells per well and further incubated for 24 h. Then, the medium in the wells was replaced with P. gingivalis at an MOI of 100, 50, or 0. After incubation for 0 h, 4 h and 6 h, 10 μL of MTT solution (Cat# M1020, MTT Cell Proliferation and Cytotoxicity Assay Kit, Solarbio) was added into each well and the plates were further incubated for 4 h. After removing the medium containing MTT, 110 μL of DMSO (Cat# M1020, Solarbio) was added into each well to dissolve the formazan crystals with low-speed shaking for 10 min. The OD490 value was measured using a microplate reader. Untreated cells (MOI 0 for 0 h) were used as controls.
9
Immunomodulatory Effects of TP5 in Mouse Splenocytes
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The TP5 was chemically synthesized by Beijing Protein Innovation Co., Ltd. (Beijing, China), and the purity surpassed 98%. Anti-mouse monoclonal antibodies of CD3-FITC, CD19-PE, CD8-FITC, and CD4-PE were provided by the BioLegend company (San Diego, CA, USA); the alginic acid (AA), Mouse Spleen NK Cells Isolation Kit, and MTT Cell Proliferation and Cytotoxicity Assay Kit were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China); the Immunoglobulin G Assay Kit, Immunoglobulin M Assay Kit, Tumor Necrosis Factor-α (TNF-α) Assay Kit, Interferon-γ (IFN-γ) Assay Kit, Interleukin-2 (IL-2) Assay Kit, and Interleukin-4 (IL-4) Assay Kit were provided by the Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu province, China). However, the other reagents used in the present paper were of analytical grade.
10
Cell Proliferation Assay Using MTT
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Cell proliferation assay was determined by MTT Cell Proliferation and Cytotoxicity Assay Kit (Solarbio, Beijing, China). Briefly, the cells were seeded in 96-well plates at 2.5 × 104 cells/cm2 (n = 6) and cultured in a growth medium for 24 h. MTT reagent (10 μL) and serum-free basal media (90 μL) were added to each well and incubated for 4 h. The supernatant was aspirated; formazan solutions (110 μL) were added to each well to dissolve the MTT formazan. Absorbance was measured at 490 nm using the Spectra Max® Plus Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA).
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