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Multitox fluor multiplex cytotoxicity assay kit

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The MultiTox-Fluor Multiplex Cytotoxicity Assay Kit is a fluorescent-based assay that measures cell viability and cytotoxicity simultaneously in the same sample. The kit employs two fluorogenic protease substrates to detect live and dead cells.

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Lab products found in correlation

Countess 2 automated cell counter, by Thermo Fisher Scientific (2 mentions) Multiplate reader, by PerkinElmer (1 mentions) Penicillin streptomycin, by Merck Group (1 mentions) Dulbecco s modified eagle medium, by Merck Group (1 mentions) Ril 2, by R&D Systems (1 mentions) Cal27 cell line, by American Type Culture Collection (1 mentions) Rpmi 1640 medium, by Merck Group (1 mentions) Penicillin streptomycin, by R&D Systems (1 mentions) Hoechst 33342, by Thermo Fisher Scientific (1 mentions) Infinite 200 pro, by Tecan (1 mentions) Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (1 mentions) Celltiter 96 aqueous one solution cell proliferation assay kit, by Promega (1 mentions)

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MultiTox-Fluor Multiplex Cytotoxicity Assay (26 citations) Cytotoxicity assay kit (19 citations) MultiTox-Fluor Cytotoxicity Assay (5 citations) MultiTox-Fluor Multiplex Cytotoxicity Kit (2 citations) Cytotoxicity assays (2 citations)

8 protocols using multitox fluor multiplex cytotoxicity assay kit

1

Cytotoxicity Assay of TDP-43 Mutants

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We estimated the cytotoxicity and viability of transfected N2a cells using the MultiTox-Fluor multiplex cytotoxicity assay kit (Promega). Briefly, N2a cells were seeded onto 96-well black culture plates and plasmids encoding FLAG-tagged WT or mutant TDP-43 and 3B12A scFv were co-transfected at 0.1 µg each per well. At 48 h after transfection, cell-permeant glycyl-phenylalanylamino fluorocoumarin (GF-AFC) and cell-impermeant bisalanyl-alanyl-phenylalanyl-rhodamine 110 (bis-AAF-R110), fluorescent indicators for live cells and dead cells, respectively, were applied. After a 2-h incubation, relative fluorescence emission was measured on a multi-plate reader (Perkin Elmer) with excitation/emission of 400/505 nm for live cells and 485/520 nm for dead cells.
Tamaki Y., Shodai A., Morimura T., Hikiami R., Minamiyama S., Ayaki T., Tooyama I., Furukawa Y., Takahashi R, & Urushitani M. (2018). Elimination of TDP-43 inclusions linked to amyotrophic lateral sclerosis by a misfolding-specific intrabody with dual proteolytic signals. Scientific Reports, 8, 6030.
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2

Cytotoxic Effects of NK Cells on HNSCC

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We used two HNSCC cell lines in this study. Cal27 cell line (tongue origin) was purchased from ATCC and was maintained in Dulbecco’s Modified Eagle Medium (Sigma) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Sigma). JHU-29 (tongue) cell line was procured from the Johns Hopkins University, and was maintained in RPMI-1640 medium (Sigma) supplemented with 10% FBS and 1% penicillin/streptomycin. The human NK cell line (NK3.3) was cultured in RPMI-1640 medium supplemented with 10% FBS, 1% glutamine, 1% penicillin-streptomycin, and 200 IU/ml recombinant IL-2 (rIL-2) (R & D Systems) (16 (link)). We added IL-12 overnight to the NK 3.3 cells, then removed residual IL-2 by washing, exposed with BME (1% v/v) for additional 20 h before incubating with cancer cells. HNSCC cells were co-cultured with BME treated NK3.3 cells at different Tumor Cell/Target: Effector Cell (T:E) (1:10) ratios for 24 hr. Cytotoxicity was measured by using a multiTox-fluor multiplex cytotoxicity assay kit (Promega) following the manufacturer’s protocol, and readings were taken using a Bio-Tek plate reader.
Bhattacharya S., Muhammad N., Steele R., Kornbluth J, & Ray R.B. (2017). Bitter Melon Enhances Natural Killer Mediated Toxicity against Head and Neck Cancer Cells. Cancer prevention research (Philadelphia, Pa.), 10(6), 337-344.
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3

Mitochondrial Function Assays

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MitoSOX Red mitochondrial superoxide indicator (Life Technologies) and Hoechst-33342 (Life Technologies) were used for mitochondrial ROS production, TMRM (Life Technologies) for mitochondrial membrane potential, ATP-based CellTiter-Glo luminescent cell viability kit (Promega) for intracellular ATP levels, MultiTox-Fluor Multiplex Cytotoxicity Assay Kit (Promega) for cytotoxicity and viability. Fluorescence and luminescence were recorded by Infinite 200 PRO (TECAN). The details of these experimental methods are described in “Supplementary Methods S1”.
Hikiami R., Morimura T., Ayaki T., Tsukiyama T., Morimura N., Kusui M., Wada H., Minamiyama S., Shodai A., Asada-Utsugi M., Muramatsu S.I., Ueki T., Takahashi R, & Urushitani M. (2022). Conformational change of RNA-helicase DHX30 by ALS/FTD-linked FUS induces mitochondrial dysfunction and cytosolic aggregates. Scientific Reports, 12, 16030.
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4

Multiplex Cytotoxicity Assay for ITO and Cr(VI) Exposure

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The MultiTox-Fluor Multiplex Cytotoxicity Assay Kit from Promega (Madison, WI) was used for cell viability assays according to the manufacturer's instructions. RAW 264.7 cells (5 × 104/well) and JB6/AP-1 cells (4 × 104/well) were grown to ~80% confluency in 96-well plates. Cells were exposed to either 50 μg/ml, 150 μg/ml or 250 μg/ml of ITO particles or 1 mM Cr (VI) as a positive control for 4 h or 24 h. The fluorogenic, cell permeant peptide substrate glycyl-phenylalanyl-aminofluorocoumarin (GF-AFC) was used to determine viability.
The dose of 50 μg/ml has previously been calculated to represent approximately 3 years of average workplace exposure, whereas a higher dose of 1 mg/ml is more representative of a career-long exposure. The following formula was used based on airborne particle concentrations of 0.1 mg/m3 at the average resting human inhaled air volume over an 8 h workday, counting 260 workdays per year:
Lung burden=respirable dust concentration×inhaled air volume/workday×alveolar deposition fraction×days.
The work done in this study therefore represents a range of exposures calculated to be at a minimum of 3 years, but less than a career long exposure (30–65 years, depending on the department within the indium plant) (Badding et al., 2014 (link)).
Olgun N.S., Morris A.M., Barber T.L., Stefaniak A.B., Kashon M.L., Schwegler-Berry D., Cummings K.J, & Leonard S.S. (2017). Comparison of the toxicity of sintered and unsintered indium-tin oxide particles in murine macrophage and epidermal cells. Toxicology and applied pharmacology, 331, 85-93.
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5

Cell Viability Assays of Transfected Cells

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Cells transfected with CYLD or pcDNA3 vectors were analysed using a tetrazolium-based colorimetric assay (3-[4,5-dimethylthiazol-2-yl]- 5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium [MTS] test) (CellTiter 96 AQueous one solution cell proliferation assay kit; Promega) or a MultiTox-fluor multiplex cytotoxicity assay kit (Promega) at 48 h after transfection.
Yamashita S., Matsuo Y., Tawara N., Hara K., Yamamoto M., Nishikami T., Kawakami K., Zhang X., Zhang Z., Doki T, & Ando Y. (2019). CYLD dysregulation in pathogenesis of sporadic inclusion body myositis. Scientific Reports, 9, 11606.
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6

Protein Toxicity Screening Assays

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To test potential protein toxicity (Additional file 1: Figure S4), we co-transfected in HeLa-JVM cells pcDNA6/myc-His B, a blasticidin S-resistance gene (bsr)-containing vector, together with empty vector (pcDNA3) or test expression constructs. On day 2, cells were expanded to T75 flasks and selection with 5 μg/ml blasticidin was begun. After 12 days, cells were fixed, stained with Giemsa and colonies were counted. Similarly, we co-transfected in HeLa cells pcDNA3, a neomycin (neo)-resistant vector, together with either empty vector (pcDNA6/myc-His B) or test expression constructs, followed by selection of cells with 500 μg/ml Geneticin (G418, Thermo Fisher).
Trypan Blue exclusion assays were performed in HEK 293T cells. Following staining, live and dead cells were counted using a Countess II Automated Cell Counter (Thermo Fisher Scientific). Use of the MultiTox-Fluor Multiplex Cytotoxicity Assay kit (Promega) followed manufacturer's instructions. This assay simultaneously measures cell viability and cytotoxicity in a single-reagent reaction, permitting ratios of live to dead cell readings to be calculated.
Pereira G.C., Sanchez L., Schaughency P.M., Rubio-Roldán A., Choi J.A., Planet E., Batra R., Turelli P., Trono D., Ostrow L.W., Ravits J., Kazazian H.H., Wheelan S.J., Heras S.R., Mayer J., García-Pérez J.L, & Goodier J.L. (2018). Properties of LINE-1 proteins and repeat element expression in the context of amyotrophic lateral sclerosis. Mobile DNA, 9, 35.
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7

Cytotoxicity Assessment of Ag-ZnO Nanorods

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Fifty thousand 3 T3 mouse fibroblast cells were seeded onto a 24-cell culture plate and incubated in a 5% CO2 incubator overnight, and samples of Ag-ZnO nanorods were incubated with cells for 24 h per well. Different amount of hybrid nanostructures (0.03, 0.07, 0.10 mg/ml) were used in the test. The control sample was cultured cells without the produced heteronanostructured samples. The MultiTox-Fluor Multiplex Cytotoxicity Assay Kit (Promega, Sunnyvale, CA, USA) is used to measure relative cell viability. The measurement process followed the protocol of the product [27 ]. The reagent was added to the 96-well plate and incubated at 37°C for 30 min. Triplicates of all samples were measured. Fluorescent signals were measured at an excitation of 400 nm and an emission of 505 nm for live cells, then at an excitation (λex) of 485 nm and an emission (λem) of 520 nm for dead cells.
Chen Y., Tse W.H., Chen L, & Zhang J. (2015). Ag nanoparticles-decorated ZnO nanorod array on a mechanical flexible substrate with enhanced optical and antimicrobial properties. Nanoscale Research Letters, 10, 106.
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8

Cell Viability and Cytotoxicity Assays

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Trypan Blue exclusion assays were performed in HEK 293T cells at 4 days post-transfection. Following staining, live and dead cells were counted using a Countess II Automated Cell Counter (Thermo Fisher Scientific). Use of the MultiTox-Fluor Multiplex Cytotoxicity Assay kit (Promega) followed manufacturer’s instructions. This assay simultaneously measures cell viability and cytotoxicity in a single-reagent reaction permitting ratios of live to dead cell readings to be calculated.
Goodier J.L., Wan H., Soares A.O., Sanchez L., Selser J.M., Pereira G.C., Karma S., García-Pérez J.L., Kazazian HH J.r, & García Cañadas M.M. (2023). ZCCHC3 is a stress granule zinc knuckle protein that strongly suppresses LINE-1 retrotransposition. PLOS Genetics, 19(7), e1010795.
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