Human ESCC cells were treated with rMV-Hu191 at the indicated time and MOI and then subjected to microscopy imaging, cell viability/death, immunoblotting analyses, or flow cytometry. Cell viability was detected using the CCK-8 assay (Targetmol, C0005). LDH release was measured to analyze cell death using the cytotoxicity assay kit (Beyotime, C0016) according to the manufacturer’s instructions. ATP levels were measured using the ATP assay kit (Beyotime, S0026). For flow cytometry analyses, Cells were collected, washed with PBS twice and stained using the Annexin V-FITC/PI Apoptosis Assay Kit (Meilunbio, MA0220) according to the manufacturer’s instructions. Stained cells were analyzed with a Beckman CytoFlex, and data were processed using FlowJo software.
Cytotoxicity assay kit
Manufactured by Beyotime
Sourced in China
The Cytotoxicity Assay Kit is a laboratory instrument designed to measure the cytotoxic effects of various compounds on cells. The kit provides a standardized method for quantifying cell viability and proliferation, allowing researchers to assess the potential toxic impact of drugs, chemicals, or other treatments on living cells.
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Lab products found in correlation
Cck 8 assay, by Targetmol (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Atp assay kit, by Beyotime (1 mentions)
Ldh cytotoxicity assay kit, by Beyotime (1 mentions)
Lactate dehydrogenase, by Beyotime (1 mentions)
Dab horseradish peroxidase color development kit, by Beyotime (1 mentions)
Cleaved caspase 3, by Proteintech (1 mentions)
Sybr green premix, by Takara Bio (1 mentions)
Alt assay kit c009 2 1, by Nanjing Jiancheng (1 mentions)
Ast assay kit c010 2 1, by Nanjing Jiancheng (1 mentions)
Peg300, by Merck Group (1 mentions)
Grp78 antibody, by Proteintech (1 mentions)
Chop antibody, by Proteintech (1 mentions)
Pe annexin 5 apoptosis detection kit, by BD (1 mentions)
Tunel apoptosis assay kit, by Beyotime (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Tween 80, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
16 protocols using cytotoxicity assay kit
1
Evaluating rMV-Hu191 Cytotoxicity in ESCC
2
ADCC Assay for Tumor Cell Lysis
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The LDH (lactate dehydrogenase) Cytotoxicity Assay Kit (Beyotime, Shanghai, China) was used to detect the capacity of PBMCs (effector cells) to trigger lysis of tumour cells in response to different drugs. Human peripheral blood mononuclear cells (PBMCs) were obtained from leukocytes, which were isolated from the peripheral blood of healthy donors. According to the guidelines of the Medical Ethical Committee of NJMU, all the donors in this study signed an informed consent. HepG2 and shMet-HepG2 cells (target cells) were seeded in 96-well plates (8 × 103 cells/well) overnight. For the ADCC assay, tumour cells (target cells) were incubated with a concentration of 10 μM anti-c-Met IgG and anti-c-Met IgG-OXA. PBMCs were incubated with tumour cells at an effector to target (E: T) ratio of 5:1, 30:1 and 100:1. After 4 h of co-incubation at 37°C, the cell supernatants were transferred to a 96-well plate, and LDH release was detected using the LDH Cytotoxicity Assay Kit (Beyotime, Shanghai, China) and quantified by measuring the absorbance at 490 nm according to the manufacturer's protocol. The cytotoxicity percentage of anti-c-Met IgG or anti-c-Met IgG-OXA was calculated as cytotoxicity% = 100 ×([experimental –effector spontaneous –target spontaneous]/[target maximum—target spontaneous]).
3
Investigating ER Stress-Mediated Apoptosis
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OEA, DMSO, PEG300, and Tween-80 were purchased from Sigma Chemical (USA). Tunicamycin (TM) was purchased from MCE (NJ, USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Viva Cell Biosciences (China). RIPA Lysis Buffer, PMSF, Cell Counting Kit-8 (C0037), DAB Horseradish Peroxidase Color Development Kit, lactate dehydrogenase (LDH), Cytotoxicity Assay Kit, and TUNEL Apoptosis Assay Kit (C1086) were purchased from Beyotime (China). PPARα antibody (Cat: 15540-1-AP), Grp78 antibody (Cat: 11587-1-AP), CHOP antibody (Cat: 15204-1-AP), Caspase 12 antibody (Cat: 55238-1-AP), cleaved-Caspase 3 (Cat: 19677-1-AP), Bcl2 (Cat: 12789-1-AP) antibody, Bax antibody (Cat: 50599-2-Ig), β-actin antibody (Cat: 66009-1-Ig), and HRP-conjugated goat anti-mouse/rabbit IgG (SA00001-1/2) were purchased from Proteintech (China). ALT assay kit (C009-2-1) and AST assay kit (C010-2-1) were purchased from Jiancheng Bioengineering Institute (Nanjing, China). Annexin V PE Apoptosis Detection Kit (559763) was purchased from BD Pharmingen (NJ, USA). Lipofectamine™3000 and TRIzol were purchased from Invitrogen. SYBR Green® Premix was purchased from TaKaRa. Chemiluminescent kit was purchased from NCM Biotech (Suzhou, China).
4
Synthesis and Evaluation of Copper-Doxorubicin Nanotherapeutics
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Chemicals. Hydroxyethyl starch (HES) with average molecular weight (Mw) 130 kDa, molar substitution of hydroxyethyl 0.4 was gift from Life Science & Technology Co., Ltd. (Wuhan, China). Doxorubicin (99%) was bought from Beijing Huafenglianbo Technology Co., Ltd. (Beijing, China). Copper (II) chloride dihydrate (CuCl2.2H2O, 99.99%), dopamine hydrochloride (98%), copper standard solution (500 ppm), methylene blue, dithiothreitol (DTT) and reduced glutathione (GSH, 98%) were purchased from Sigma-Aldrich. Ammonia solution (NH3.H2O, 25-28%), methanol, dimethyl sulfoxide (DMSO), tween-80 and hydrogen peroxide (H2O2) were purchased from SinopharmChemical Reagent Co.Ltd, (Shanghai, China). Ultrapure water (Millipore Milli-Q grade, 18.2 MΩ) was used in all the experiments. All chemicals were analytical grade and used directly without further purification.
Biological reagents . GSH and GSSG Assay Kit and Cytotoxicity Assay Kit were bought from Beyotime. Annexin V-APC/7-AAD apoptosis kit was purchased from Multisciences (LianKe) Biotech, Co., Ltd. 2, 7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA), Cell Counting Kit-8 (CCK-8) and Calcein-AM/PI double stain kit was purchased from Yeasen Biotechnology Co., Ltd. RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Gibco BRL/Life Technologies, Grand Island, NY, USA.
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5
Cytotoxicity Quantification via LDH Assay
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Cytotoxicity was quantified by assessing the release levels of LDH using a cytotoxicity assay kit (Beyotime Biotech). Briefly, CEFs seeded in 96-well plates were exposed to NDV at an MOI of 1 for the designated time. Concurrently, control groups were established, including the blank background group, the control sample group, and the maximum enzyme activity group. The reagent to detect LDH release was added to the wells and allowed to incubate for 1 h prior to the commencement of testing. The medium was then collected and centrifuged to remove any cell debris. Then, we added 60 µL of LDH test solution to the supernatant for 30 min (min) at room temperature in the absence of light. Finally, the optical density was determined at 490 nm. The percentage of cytotoxicity was calculated in accordance with the manufacturer’s guidelines.
6
Lipase-Catalyzed Polymer Synthesis
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PTMC was purchased from Shanghai Leon Chemical Co., Ltd (Shanghai, China). VCM-HCl was supplied by Dalian Meilun Biology Technology Co., Ltd (Dalian, China). Lipase solution from Thermomyces lanuginosus (100,000 U/g) was obtained from Sigma-Aldrich (St Louis, MO, USA) and used as received. Tetrahydrofuran (THF) and anhydrous hexane were purchased from Tianjin Shield Specialty Chemical Co., Ltd (Zhejiang, China). Beyotime Biotechnology Ltd Co (Shanghai, China) supplied the Cytotoxicity Assay Kit and all cell culture reagents. Methanol (HPLC grade) was purchased from Fisher Chemical (Suzhou, China). Other reagents and chemicals were of analytical reagent grade, unless indicated otherwise.
7
Cytotoxicity Assay of AGS Cells
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The cytotoxicity of AGS cells were assessed by measurement of lactate dehydrogenase (LDH) expression in the cultured supernatants using Cytotoxicity Assay Kit (Beyotime, Wuhan, Hubei Province, China). The low level control was defined as LDH expression in the supernatant of AGS cells, while the high level control was defined as LDH expression in the supernatant of Triton X-100 treated AGS cells. The percentage of target cell death was calculated using the following equation: (experiment value − low level control)/(high level control − low level control) × 100% [17 (link)–20 (link)].
8
Cell Viability and Colony Formation Assay
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Thiazolyl blue tetrazolium bromide (MTT) assay: Cytotoxicity Assay Kit (Beyotime, Beijing, China) was used to monitor cell viability. CRC cells transfected with inhibited or overexpressed ZEB1-AS1 were placed on 96-well plates with six replicate wells (3000 cells/ well). Colony formation assay: About 800 cells were put in six-well plates and cultured in media with 10% FBS for 2 weeks. Finally, these cells were fixed by 4% paraformaldehyde and stained by 0.1% crystal violet (Beyotime). There appeared visible colonies that could be counted. Experiments were repeated for three times and performed in accordance with the manufacturer's recommendations.
9
LDH Cytotoxicity Assay Protocol
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Cytotoxicity assay was determined by LDH (lactate dehydrogenase) Cytotoxicity Assay Kit (Beyotime Biotechnology, Jiangsu, China) according to the manufacturer’s protocol. In brief, 100 μL cell supernatants from each well were collected and transferred to a new 96-well plate. 50 μL LDH reagent was added to each well and then incubated at 37 °C for 30 min protected from light. The OD values were measured at 490 nm by a Synergy HT Microplate Reader. The cytotoxicity was calculated by the relative OD values to the control group.
10
Cytotoxicity Measurement by LDH Assay
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Cytotoxicity measurement was analyzed by LDH (lactate dehydrogenase) Cytotoxicity Assay Kit (C0017, Beyotime Biotechnology, China). T cells specifically induced for 14 days were collected, and tumor cells were added into the 96-well plate at a certain experimental proportion for a 6-h cell killing experiment. After 6 h, the 96-well plate was gently shaken to ensure the uniform distribution of LDH in the supernatant. The 96-well plates were centrifuged at room temperature at 600 g for 10 min, then 10 μL of supernatant was transferred from each plate to the new 96-well plate, and 100 μL of the LDH mixture was added to each well for 30 min incubation at room temperature. Finally, the absorbance value was measured at 450 nm (reference wavelength was 650 nm) in the enzyme plate analyzer. The killing ratio was calculated as follows:
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