Cytotox green

Manufactured by Sartorius

Sourced in United States

Cytotox Green is a fluorescent dye used for the detection and quantification of cell death in cell-based assays. It is a sensitive and reliable tool for measuring cytotoxicity and monitoring cell viability.

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Lab products found in correlation

Cytolight red, by Sartorius (2 mentions) Incucyte s3, by Sartorius (2 mentions) Marimastat, by Santa Cruz Biotechnology (2 mentions) Cellrox green, by Thermo Fisher Scientific (2 mentions) Mg 132, by Promega (2 mentions) Px ethyl, by Merck Group (2 mentions) Cy and alexa fluor conjugated secondary antibodies, by Jackson ImmunoResearch (2 mentions) Z vad fmk, by Adooq (2 mentions) Incucyte live cell analysis system, by Sartorius (1 mentions) Celltracker, by Thermo Fisher Scientific (1 mentions) Imagelock plate, by Sartorius (1 mentions) Imagexpress pico, by Molecular Devices (1 mentions) Hoechst stain, by Merck Group (1 mentions) Prism 7.0c, by GraphPad (1 mentions) Incucyte, by Sartorius (1 mentions) Incucyte s3 live cell analysis system, by Satorius (1 mentions) Cytotox green dye, by Sartorius (1 mentions) Nigericin, by Cayman Chemical (1 mentions) Incucyte s3 live cell imager, by Sartorius (1 mentions) Incucyte device, by Sartorius (1 mentions)

12 protocols using cytotox green

Live Neuronal Culture Cytotoxicity Assay

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Live neuronal cultures were incubated with 0.25 μM Cytotox Green (catalog # 4633, Essen Bioscience, Ann Arbor, MI) and 1 μM Cytolight red (catalog # 4706, Essen Bioscience, Ann Arbor, MI) reagents for 20 min and imaged in an IncuCyte S3 (Essen Bioscience, Ann Arbor, MI) with a 20X objective. Cell counts were obtained from images in 9 fields collected for every well, in bright-field to capture all cells and fluorescence for Cytotox Green to identify dead cells.

Hughes R.O., Bosanac T., Mao X., Engber T.M., DiAntonio A., Milbrandt J., Devraj R, & Krauss R. (2021). Small Molecule SARM1 Inhibitors Recapitulate the SARM1−/− Phenotype and Allow Recovery of a Metastable Pool of Axons Fated to Degenerate. Cell reports, 34(1), 108588.

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Immunocytochemistry Reagents and Protocols

Cited 3 times

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Mouse anti-VE-cadherin antibody (sc-9989) diluted 1:50 was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Cy and Alexa Fluor-conjugated secondary antibodies were acquired from Jackson Immunoresearch (Philadelphia, PA, USA) and Molecular Probes (Eugene, OR, USA), respectively, and used for immunocytochemistry. HIF2α inhibitor [51 (link)] (Axon 2034) was obtained from Axon Medchem (Hanzeplein, The Netherlands). Z-VAD-FMK was obtained from Adooq-bioscience (187389-52-2, Irvine, CA, USA) and Santa Cruz Biotechnology (sc-3067, Dallas, TX, USA). Marimastat from Santa Cruz Biotechnology (sc-202223, Dallas, TX, USA), MG-132 from Promega (G9951, Madison, WI, USA). Cytotoxgreen was obtained from Essen BioScience (Ann Arbor, MI, USA) and CellROXgreen from Molecular Probes (Eugene, OR, USA). PX-ethyl was purchased from Sigma (St. Louis, MO, USA). According to the Sigma safety data sheet, safety measures of eye shields, face shields, full-face respirator, and gloves should be taken. PX was used according to our previously reported protocol [43 (link),44 (link)] All other reagents applied in this study were used in accordance to the known literature and the supplier’s guidelines.

Rand D., Ravid O., Atrakchi D., Israelov H., Bresler Y., Shemesh C., Omesi L., Liraz-Zaltsman S., Gosselet F., Maskrey T.S., Schnaider Beeri M., Wipf P, & Cooper I. (2021). Endothelial Iron Homeostasis Regulates Blood-Brain Barrier Integrity via the HIF2α—Ve-Cadherin Pathway. Pharmaceutics, 13(3), 311.

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Live Neuronal Culture Cytotoxicity Assay

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2D Co-culture Assay for Endothelial-Tumor Interactions

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For 2D coculture experiments, 10000 MCF10 or MCF10A-NeuN cells were seeded on confluent CellTracker (Invitrogen) labeled HUVEC monolayers, with monocultures as controls. After 24 h, cells were fixed in 4% paraformaldehyde and prepared for confocal microscopy as described below. 2D cocultures were also monitored over time using the IncuCyte Live Cell Analysis System (Essen Bioscience, Ann Arbor, MI). To fluorescently distinguish the two different cell types, HUVEC were stably transfected to label mitochondria with green fluorescent protein (GFP-HUVEC; virus kindly provided by Dr. Christian Sells, Drexel College of Medicine) or the MDA-MB-231 breast cancer cell line was stably transfected with red fluorescent protein using Nuclight Red (RFP-MDA-MB-231; Essen Bioscience). Samples included (1) confluent GFP-HUVEC; (2) 10000 RFP-MDA-MB-231; and (3) confluent GFP-HUVEC with 10000 RFP-MDA-MB-231. In addition, HUVEC death was measured over time with a cytotoxicity assay. Samples included (1) confluent HUVEC; (2) 10000 MDA-MB-231; and (3) confluent HUVEC with 10000 MDA-MB-231. Cytotox Green (Essen Bioscience) was added to these samples to quantify dead cells. Samples were imaged every 4 h for 3 days. Images were analyzed using Incucyte Zoom software.

Swaminathan S., Ngo O., Basehore S, & Clyne A.M. (2017). Vascular Endothelial–Breast Epithelial Cell Coculture Model Created from 3D Cell Structures. ACS biomaterials science & engineering, 3(11), 2999-3006.

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Wound Healing Assay with JIMT Cells

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JIMT cells were plated at a density of 38,000 cells/well into a 96-well ImageLock plate (Essen Biosciences) to achieve confluence within 24 h. The following day, the WoundMaker tool was used to scratch the cell monolayer. The cells were washed twice with growth media and then fresh growth media (vehicle) or growth media containing drug were added to the cells in the presence of Cytotox Green (4663, Essen Bioscience) reagent to measure cell viability throughout the wound healing process. Drug doses were based on isobole testing and were selected to be minimally toxic for the duration of the assay time. The plate was imaged every four hours to calculate the wound width. The wound size was normalized to the initial wound width (t = 0 h) and then normalized relative to the untreated, vehicle control.

Krutilina R.I., Hartman K.L., Oluwalana D., Playa H.C., Parke D.N., Chen H., Miller D.D., Li W, & Seagroves T.N. (2022). Sabizabulin, a Potent Orally Bioavailable Colchicine Binding Site Agent, Suppresses HER2+ Breast Cancer and Metastasis. Cancers, 14(21), 5336.

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Cytotoxicity Assay with Imaging

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Cells were seeded in 96-well plates and incubated with inhibitors at their respective experimental concentrations (indicated above) along with the membrane-impermeable nuclear dye Cytotox Green (Essen BioScience, Inc. Ann Arbor, MI) and cell-permeable Hoechst stain (Sigma). Cytotoxicity was measured on an ImageXpress Pico (Molecular Devices) by quantifying the fraction of Cytotox Green-positive, permeable cells among total cells.

Ramanathan H.N., Zhang S., Douam F., Mar K.B., Chang J., Yang P.L., Schoggins J.W., Ploss A, & Lindenbach B.D. (2020). A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating. mBio, 11(2), e00467-20.

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Quantifying Viral Infection in Neuronal Cells

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We coated 96-well plates with fibronectin as previously described and seeded 5 × 10⁴ SH-SY5Y cells or 2 × 10⁴ hNSCs per well. The next day, cells were inoculated with each virus in triplicate at multiplicities of infection (MOIs) of 0.1 and 0.01 for SH-SY5Y cells and 0.01 and 0.001 for hNSCs. We added Cytotox Green (Essen Bioscience, https://www.essenbioscience.com) for a final well volume of 100 μL and a final concentration of 250 nM. Cells were then imaged with an IncuCyte (Essen Bioscience) by taking 3 images per well with the 20× objective every 3 hours during a 97-hour time course. We measured confluence and fluorescent intensity using IncuCyte S3 software. We performed all statistical analyses using Prism 7.0c (https://www.graphpad.com).

Evans A.B., Winkler C.W, & Peterson K.E. (2019). Differences in Neuropathogenesis of Encephalitic California Serogroup Viruses. Emerging Infectious Diseases, 25(4), 728-738.

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Immunocytochemistry with VE-cadherin antibody

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Mouse anti-VE-cadherin antibody (sc-6458) diluted 1:50 was obtained from Santa Cruz Biotechnology (United States). Cy and Alexa Fluor-conjugated secondary antibodies were acquired from Jackson Immunoresearch (United States) and Molecular Probes (United States), respectively, and used for immunocytochemistry. Z-VAD-FMK was obtained from Adooq-bioscience (187389-52-2, United States) and Santa Cruz Biotechnology (sc-3067, United States). Marimastat from Santa Cruz Biotechnology (sc-202223, United States), MG-132 from Promega (G9951, United States). Cytotoxgreen was obtained from Essen BioScience (United States) and CellROXgreen from Molecular Probes (United States). PX-ethyl was purchased from Sigma (United States), according to Sigma safety data sheet safety measures of eyeshields, face shields, full-face respirator and Gloves should be taken. For assessing the BBB response, PX freshly made in ethanol to 400 mM stock solution was immediately diluted in the medium to the desired final concentrations and added to the cell culture. All other reagents applied in this study were used in accordance to the known literature and the supplier's guidelines.

Rand D., Ravid O., Atrakchi D., Israelov H., Bresler Y., Shemesh C., Omesi L., Liraz‐Zaltsman S., Gosselet F., Maskrey T.S., Beeri M.S., Wipf P., & Cooper I. (2020). Endothelial iron homeostasis regulates BBB integrity via the HIF2α – Ve-cadherin pathway.

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Quantifying Neutrophil Extracellular Trap (NET) Formation

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A total of 20,000 neutrophils were seeded to 96‐well plates (Nunc, Thermo Fischer Scientific) in 50 μL RPMI without phenol red containing Cytotox Green dye (Sartorius; final concentration of 50 nM). The plate was incubated for 15 min at 37°C to allow cell adherence. The cells were then stimulated with 50 μL RPMI containing serum (final amount of 10%) from patients with EWS or from pediatric controls and placed in the IncuCyte S3 Live‐Cell Analysis System (37°C, 5% CO2; Satorius). Four pictures per condition were taken every 30 min with a 20× objective for a period of 3 h. Since the fluorescent dye Cytotox Green (Sartorius) only binds extracellular DNA, NET formation could be studied over time by measuring the increase in fluorescent particles. The images were processed by the basic analyzer unit of the IncuCyte S3 2021 Software (Sartorius). The proportion of neutrophils producing NETs was calculated by dividing the number of green count (green channel) by the number of cells (phase channel) and expressed as a percentage.

Shukrun R., Baron S., Fidel V., Shusterman A., Sher O., Kollender N., Levin D., Peled Y., Gortzak Y., Ben‐Shahar Y., Caspi R., Gordon S., Manisterski M, & Elhasid R. (2023). Suggested role for neutrophil extracellular trap formation in Ewing sarcoma immune microenvironment. Cancer Science, 115(1), 36-47.

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Quantification of Pyroptosis in RAW 264.7 Cells

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To perform pyroptosis assays, 30,000 RAW 264.7 cells (WT and Hprt KOs) per well were plated in 96-well plates, in either regular culture media (un-primed control) or media containing LPS+IFNy, and incubated for 24 hours. Three hours before induction of pyroptosis, media was replaced with the addition of 250nM Cytotox green (Sartorius). Pyroptosis was induced with 10μM nigericin (Cayman Chemical: 11437) and the cells were subsequently imaged over a time course using an Incucyte S3 Live-Cell imager (Sartorius). Pyroptosis was measured based on a cell-by-cell classification analysis that quantified the percentage of green positive cells out of the total number of cells imaged.

John S.V., Seim G.L., Erazo-Flores B.J., Steill J., Freeman J., Votava J.A., Arp N.L., Qing X., Stewart R., Knoll L.J, & Fan J. (2023). Macrophages undergo functionally significant reprograming of nucleotide metabolism upon classical activation. bioRxiv.

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