Wst 1 cell proliferation and cytotoxicity assay kit

Manufactured by Beyotime

Sourced in China

The WST-1 Cell Proliferation and Cytotoxicity Assay Kit is a colorimetric assay used to measure cell proliferation, cell viability, and cytotoxicity. It utilizes the water-soluble tetrazolium salt WST-1 as the key component.

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WST-1 Assay Kit WST-1 assay Cytotoxicity Assay Kit WST-1 kit Cell Proliferation Kit WST-1

50 protocols using wst 1 cell proliferation and cytotoxicity assay kit

Prx and LIG Preparation for Cell Studies

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Recombinant mouse Prxs were purchased commercially: Prx1 and Prx5 (Batch No: 50552-mM08E and 50551-M07E) from Sino Biological (Beijing, China), Prx2–Prx4 and Prx6 (Batch No: RPF757Mu01, RPF753Mu01, RPF754Mu01 and RPF756Mu01) from USCN Life Science (Wuhan, Hubei, China). The primary antibodies used in this study, including iNOS, TLR4 and NF-κB p65, were purchased from Boster Biological Technology (Wuhan, Hubei, China), Santa Cruz Biotechnology (Heidelberg, Germany), and Zhongshan Jinqiao Biology (Beijing, China), respectively. WST-1 Cell Proliferation and Cytotoxicity Assay Kit were purchased from Beyotime Biological Technology (Haimen, Jiangsu, China).
Prx1–Prx6 were prepared as a 10 μM stock solution in sterile water following the manufacturer's recommended procedure, while LIG (purity > 98.5%, S1 Fig), prepared by a well-established procedure in our laboratory (Kuang et al., 2008), was dissolved in DMSO as a 50 mM stock solution. Both Prx and LIG stock solutions were kept at -80°C, and diluted with cell culture medium before use. A final concentration of 0.1% DMSO was used as vehicle for the treatment of the control cultures.

Zhao L.X., Du J.R., Zhou H.J., Liu D.L., Gu M.X, & Long F.Y. (2016). Differences in Proinflammatory Property of Six Subtypes of Peroxiredoxins and Anti-Inflammatory Effect of Ligustilide in Macrophages. PLoS ONE, 11(10), e0164586.

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CRC Cell Viability Assay by WST-1

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The CRC cell viability was measured by WST-1 Cell Proliferation and Cytotoxicity Assay Kit (Beyotime Biotechnology, Shanghai, China), as previously described 22 (link). WST-1 was added, and cells were incubated for 2 h at 37°C, and the absorbance at 450 nm was measured on an automated microplate spectrophotometer (BioTek Instruments, Winooski, VT).

Hu H.F., Xu W.W., Li Y.J., He Y., Zhang W.X., Liao L., Zhang Q.H., Han L., Yin X.F., Zhao X.X., Pan Y.L., Li B, & He Q.Y. (2021). Anti-allergic drug azelastine suppresses colon tumorigenesis by directly targeting ARF1 to inhibit IQGAP1-ERK-Drp1-mediated mitochondrial fission. Theranostics, 11(4), 1828-1844.

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Isolation and Characterization of Intestinal Epithelial Cells

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DMEM/F12 and FBS were purchased from Gibco. EGF, ITS-G and Lipofectamine Plus were products of Invitrogen. The WST-1 Cell Proliferation and Cytotoxicity Assay Kit was obtained from Beyotime, Shanghai, China. The Annexin V-FITC Apoptosis Detection Kit was a product of Calbiochem, Darmstadt, Germany. The TRAP-silver staining Telomerase Detection Kit was purchased from KeyGEN Biotech, Nanjing, China. The following primary antibodies were used: Pan-cytokeratin (clone AE1/AE3, AbD Serotec, Oxford, UK), mouse anti-Cytokeratin 18 (clone CY90, Sigma, St. Louis, MO, USA), rabbit anti-Sucrase-isomaltase (clone H-123, Santa Cruz, Heidelberg, Germany), rabbit anti-E-cadherin antibody (Genscript), mouse anti-OCLN (Clones 1G, AbD, Oxford, UK), rabbit anti-villin and anti-ZO-1 (Bioss, Beijing, China).
Healthy unsuckled 1-day-old Landrace piglets were purchased from the pig farm of Northwest Agriculture & Forestry University.
Nude mice at 4 weeks old of age were purchased from the Experimental Animal Center of The Fourth Military Medical University (Xi'an, China) and maintained in pathogen-free conditions.

Wang J., Hu G., Lin Z., He L., Xu L, & Zhang Y. (2014). Characteristic and Functional Analysis of a Newly Established Porcine Small Intestinal Epithelial Cell Line. PLoS ONE, 9(10), e110916.

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Apoptosis and Proliferation Analysis

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Apoptosis was evaluated via fluorescence-activated cell sorting after annexin V-FITC and propidium iodide staining. Adherent cells were detached with 2.5% trypsin-EDTA and added to the culture supernatant. Cells were pelleted by centrifugation at 1000 rpm for 5 min, suspended in 300 μL 1× binding buffer (FITC Annexin V Apoptosis Detection Kit I, BD Biosciences, USA) and incubated for 20 min with Annexin V-FITC (5 μL) and propidium iodide (5 μL). Flow cytometry was performed on a CytoFLEX flow cytometer, and data analyzed using CytExpert (Beckman Coulter, USA).
Cell proliferation was quantified using the WST-1 Cell Proliferation and Cytotoxicity Assay Kit (Beyotime, Shanghai, China) according to the manufacturerʹs instructions. Electronic coupling agent (1 mL) was added into WST-1, and 10 μL WST-1 solution used for every 100 μL culture medium.

Guo W., Lian S., Zhen L., Zang S., Chen Y., Lang L., Xu B., Guo J., Ji H., Wang J., Fu S., Zhang L, & Yang H. (2018). The Favored Mechanism for Coping with Acute Cold Stress: Upregulation of miR-210 in Rats. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 46(5).

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Cell Viability Assay with DHCP and TRAIL

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The WST-1 cell proliferation and cytotoxicity assay kit (C0035, Beyotime) was used to measure cell viability. Cells were treated with different concentrations of DHCP, TRAIL, or their combination, for 24 h. The medium was removed and the WST-1 solution was added, and absorbance was measured at 450 nm using a microplate reader (TECAN, infinite F50). All experiments were performed in triplicate and repeated at least three times. Cell viability under each condition is expressed as a percentage of the control set at 100%.

Chen L., Hao M., Yan J., Sun L., Tai G., Cheng H, & Zhou Y. (2021). Citrus-derived DHCP inhibits mitochondrial complex II to enhance TRAIL sensitivity via ROS-induced DR5 upregulation. The Journal of Biological Chemistry, 296, 100515.

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Cell Viability Assay Protocol

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Cells were seeded into a 96‐well plate with 3000 cells per well, and different concentrations of drugs were added after 24 h. Cell viability was measured by a WST‐1 Cell Proliferation and Cytotoxicity Assay Kit (Beyotime, Shanghai, China).^[²⁵^] The absorbance was measured at 450 nm with a microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA).

Zheng C., Liao L., Liu Y., Yang Y., He Y., Zhang G., Li S., Liu T., Xu W.W, & Li B. (2022). Blockade of Nuclear β‐Catenin Signaling via Direct Targeting of RanBP3 with NU2058 Induces Cell Senescence to Suppress Colorectal Tumorigenesis. Advanced Science, 9(34), 2202528.

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WST-1 Assay for Oxypurinol-Mediated HCC Proliferation

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A WST-1 Cell Proliferation and Cytotoxicity Assay Kit (Beyotime Institute of Biotechnology, China) was used to detect HCC cell proliferation, as described in our previous report.^{52 (link)} In brief, the abovementioned cells were seeded in 96-well culture plates at a density of 2000 cells/well. To evaluate the effects of oxypurinol (Sigma-Aldrich, Co. LLC., Shanghai, China, cat. no. O6881), a potent xanthine oxidase inhibitor,^{53 (link)} on cell proliferation, we incubated the cells with or without 50 μmol/l (μM) oxypurinol. Cell proliferation was monitored over a 72-h time period and measured according to the manufacturer’s instruction. All experiments were performed at least three times and in triplicate.

Chen G.L., Ye T., Chen H.L., Zhao Z.Y., Tang W.Q., Wang L.S, & Xia J.L. (2017). Xanthine dehydrogenase downregulation promotes TGFβ signaling and cancer stem cell-related gene expression in hepatocellular carcinoma. Oncogenesis, 6(9), e382-.

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Cell Viability Assay Using WST-1

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Cell viability was measured using a WST-1 Cell Proliferation and Cytotoxicity Assay Kit (Beyotime Biotechnology, Shanghai, China). Briefly, the cells were seeded in 96-well plates at an approximate density of 2000 cells were seeded per well. At the indicated time points, 10 μl of WST-1 reagent was added to the wells. The cells were subsequently incubated for 4 h at 37 °C. The absorbance was measured at a wavelength of 450 nm.

Zhong Y., Yang J., Xu W.W., Wang Y., Zheng C.C., Li B, & He Q.Y. (2017). KCTD12 promotes tumorigenesis by facilitating CDC25B/CDK1/Aurora A-dependent G2/M transition. Oncogene, 36(44), 6177-6189.

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Cisplatin Cytotoxicity Assay in HK-2 Cells

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The cytotoxicity of cisplatin was determined by the water-soluble tetrazolium- (WST-) 1 method using the WST-1 cell proliferation and cytotoxicity assay kit (Beyotime Biotechnology). Briefly, HK-2 were seeded in 96-well plates at 5000 cells/well and incubated at 37°C for 24 h. The media were replaced with normal media in the absence or presence of DMY (25 or 50 μM). After incubation for 24 h, cells were exposed to cisplatin (10, 20, 30, or 50 μM) for another 24 h. Then WST-1 reagent was added for 4 h at 37°C, and absorbance was measured at 450 nm wavelength using an automated microplate reader.

Wu F., Li Y., Song H., Zhang Y., Zhang Y., Jiang M., Wang F., Mu Q., Zhang W., Li L, & Tang D. (2016). Preventive Effect of Dihydromyricetin against Cisplatin-Induced Nephrotoxicity In Vitro and In Vivo. Evidence-based Complementary and Alternative Medicine : eCAM, 2016, 7937385.

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Evaluating GCV Cytotoxicity in TK-Expressing Cells

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HEK293 cells were transfected with different TK constructs in the absence of phiC31 integrase. Transfected cells were determined by GFP expression and sorted by FACS performed at 48 h after transfection. The sorted GFP-positive cells were seeded in 96-well plates at a cell density of 5.0 × 10³ cells/well. GCV concentrations (0, 0.01, 0.1, 1, 10, and 20 μg/mL) were added to the transfected cells. After 4 d, the killing effect of GCV was measured using a WST-1 cell proliferation and cytotoxicity assay kit (Beyotime) according to the manufacturer's instructions.

Yu Y., Tong Q., Li Z., Tian J., Wang Y., Su F., Wang Y., Liu J, & Zhang Y. (2014). Improved site-specific recombinase-based method to produce selectable marker- and vector-backbone-free transgenic cells. Scientific Reports, 4, 4240.

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