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Celltiter glo luminescent cell viability reagent

Manufactured by Promega
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The CellTiter-Glo luminescent cell viability reagent is a laboratory product that measures the amount of ATP present in a cell culture, which is an indicator of metabolically active cells. The reagent provides a quantitative measure of cell viability and proliferation in a simple, homogeneous assay format.

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Lab products found in correlation

Colorimetric assay kit, by Merck Group (3 mentions) Glutamine glutamate glo assay kit, by Promega (2 mentions) Penicillin streptomycin mix, by Thermo Fisher Scientific (1 mentions) Eagle s minimal essential medium emem, by Lonza (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Dimethyl sulfoxide (dmso), by AppliChem (1 mentions) Dulbecco s modified eagle medium dmem, by Lonza (1 mentions) Porcine liver esterase, by Merck Group (1 mentions) Synergy h1 multi mode reader, by Agilent Technologies (1 mentions) Infinite m1000, by Tecan (1 mentions) Fitc annexin 5 apoptosis detection kit with 7 aad, by BioLegend (1 mentions) Janus automated workstation, by PerkinElmer (1 mentions) Hoechst 33342, by Merck Group (1 mentions) Synergy h1 hybrid reader, by Agilent Technologies (1 mentions) Blasticidin, by Thermo Fisher Scientific (1 mentions) Polybrene, by Merck Group (1 mentions) Envision, by PerkinElmer (1 mentions) Flexstation 3 microplate reader, by Molecular Devices (1 mentions) Envision multilabel reader, by PerkinElmer (1 mentions)

18 protocols using celltiter glo luminescent cell viability reagent

1

Synthesis and Characterization of FA2-dPEG-DOX2

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FA2-dPEG-DOX2 was synthesized as shown in Figure 1 and was characterized by NMR spectroscopy. The details will be published elsewhere.
DOX powder-control; dimethyl-sulfoxide (DMSO) (Applichem GmbH, Darmstadt, Germany), MDA-MB-231 triple-negative human breast cancer cell line (ECACC, Salisbury, UK); Dulbecco’s Modified Eagle Medium (DMEM) (Lonza, Basel, Switzerland), Eagle’s Minimal Essential Medium (EMEM) (Lonza, Basel, Switzerland), fetal bovine serum (FBS) (Gibco, Amarillo, TX, USA), L-glutamine (Gibco, Amarillo, TX, USA), penicillin-streptomycin mix (Gibco, Amarillo, TX, USA), Hoechst 33342, Trihydrochloride, Trihydrate solution (10 mg/mL, Thermo Scientific, Waltham, MA, USA), CellTiter Glo Luminescent Cell Viability Reagent (Promega, Madison, WI, USA).
Nagy K.S., Toth K., Pallinger E., Takacs A., Kohidai L., Jedlovszky-Hajdu A., Mathe D., Kovacs N., Veres D.S., Szigeti K., Molnar K., Krisch E, & Puskas J.E. (2021). Folate-Targeted Monodisperse PEG-Based Conjugates Made by Chemo-Enzymatic Methods for Cancer Diagnosis and Treatment. International Journal of Molecular Sciences, 22(19), 10347.
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2

NMUr Cytotoxicity Assay Protocol

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Cells were seeded 100 000 cells/well in 6-well plates (total volume: 2 mL) 24 h before they were exposed to NMUr for 1 h (0, 5, 10, 20, 40, 50, 75, 100, or 200 μM). At 1 h, porcine liver esterase (1 unit/well, Sigma-Aldrich, St. Louis, MO) was added to inactivate any extracellular NMUr. This step removed excess NMUr in order to reduce variation between experiment replicates. Each treatment was performed in duplicate. After 48 h, three 50-μL aliquots were removed from each well and combined with 50 μL of CellTiter-Glo Luminescent Cell Viability reagent (Promega Corporation, Madison, WI) in 96-well plates and incubated for 20 min on a shaker. The plates were analyzed using a Synergy H1Multi-Mode Reader (BioTek, Winooski, VT) operating in the luminescent mode with an acquisition gain of 134 according to manufacturer’s instructions. Cell survival was normalized to the luminescence of the cells treated with 0 μM NMUr. IC20, IC50, and IC80 values were determined using the Gen5Microplate Reader and Imager Software. Seventeen cell lines were repeated at a different time to determine the technical variation of the assay. The IC20 values varied by 13% ± 13%, and the IC50 values varied by 10% ± 10%.
Peterson L.A., Ignatovich I.V., Grill A.E., Beauchamp A., Ho Y.Y., DiLernia A.S, & Zhang L. (2019). Individual Differences in the Response of Human β-Lymphoblastoid Cells to the Cytotoxic, Mutagenic, and DNA-Damaging Effects of a DNA Methylating Agent, N-Methylnitrosourethane. Chemical research in toxicology, 32(11), 2214-2226.
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3

Cell Viability Assay with Growth Factors

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Cell lines were obtained from the Genentech cell line repository and maintained in RPMI 1640 medium supplemented with 10% FBS and 2 mM l-glutamine. Compounds were obtained from the Genentech compound management as 10 mM dimethyl sulfoxide stock solutions. For cell viability assays, cells were plated in normal growth medium at 1000–2000 cells per well in 384-well clear-bottom black plates. The following day, compounds were serially diluted starting at indicated concentrations, then added to cells in quadruplicates. Ninety-six hours following compound addition, CellTiter-Glo Luminescent Cell Viability reagent (Promega) was added per manufacturer’s protocol. For studies with growth factors, 20 nM TGF-alpha, 100 ng/mL HGF, or 10 ng/mL FGF was added during plating of cells and at time of drug treatment.
Kirouac D.C., Schaefer G., Chan J., Merchant M., Orr C., Huang S.M., Moffat J., Liu L., Gadkar K, & Ramanujan S. (2017). Clinical responses to ERK inhibition in BRAFV600E-mutant colorectal cancer predicted using a computational model. NPJ Systems Biology and Applications, 3, 14.
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4

Cell Viability Assay with Drug Screening

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Approximately 10,000 cells were seeded per well into a 96-well plate 24 h prior to drug treatment. Drugs were threefold diluted in DMSO and kept at 1% (v/v) across all drug concentrations and control. Each drug concentration was tested in triplicate. The viability of cells were assayed using CellTiter-Glo luminescent cell viability reagent (Promega, cat. no. G7572). The luminescence signals were detected using TECAN Infinite M1000 pro multi-mode plate reader using an integration time of 250 ms. The relative luminescence units from treated wells were normalized against DMSO control wells and expressed as percentage cell viability. IC50 values were calculated using GraphPad Prism software.
Chia S., Low J.L., Zhang X., Kwang X.L., Chong F.T., Sharma A., Bertrand D., Toh S.Y., Leong H.S., Thangavelu M.T., Hwang J.S., Lim K.H., Skanthakumar T., Tan H.K., Su Y., Hui Choo S., Hentze H., Tan I.B., Lezhava A., Tan P., Tan D.S., Periyasamy G., Koh J.L., Gopalakrishna Iyer N, & DasGupta R. (2017). Phenotype-driven precision oncology as a guide for clinical decisions one patient at a time. Nature Communications, 8, 435.
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5

Proliferation Assays and Cell Cycle Analysis

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Proliferation assays with ruxolitinib and ALRN-6924 were performed in a 384-well format, using a JANUS Automated Workstation (PerkinElmer) and CellTiter-Glo Luminescent Cell Viability reagent (Promega) at 0 h and 72 h according to the manufacturer’s protocol. Each data point represents quadruplicates and experiments were repeated at least twice. IC50 values were calculated, using the GR calculator at www.grcalculator.org. Proliferation assays with lenalidomide were performed in triplicate and performed twice, with a concentration of 1 μM vs DMSO, cells were stained with PI, counted and medium was replaced every 48 h. Statistical significance was assessed by two way ANOVA and Bonferroni post-test. Detection of apoptosis was performed by Annexin V staining using the biolegend® FITC Annexin V Apoptosis Detection Kit with 7-AAD. Cell cycle analysis were performed with Hoechst33342 from SigmaAldrich® according to the manufacturer’s protocol.
Ng S.Y., Yoshida N., Christie A.L., Ghandi M., Dharia N.V., Dempster J., Murakami M., Shigemori K., Morrow S.N., Van Scoyk A., Cordero N.A., Stevenson K.E., Puligandla M., Haas B., Lo C., Meyers R., Gao G., Cherniack A., Louissaint A J.r., Nardi V., Thorner A.R., Long H., Qiu X., Morgan E.A., Dorfman D.M., Fiore D., Jang J., Epstein A.L., Dogan A., Zhang Y., Horwitz S.M., Jacobsen E.D., Santiago S., Ren J.G., Guerlavais V., Annis D.A., Aivado M., Saleh M.N., Mehta A., Tsherniak A., Root D., Vazquez F., Hahn W.C., Inghirami G., Aster J.C., Weinstock D.M, & Koch R. (2018). Targetable vulnerabilities in T- and NK-cell lymphomas identified through preclinical models. Nature Communications, 9, 2024.
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6

Cell Viability Assay with Virus

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Dissociated cells were seeded into 96-well plates, serially diluted viruses were added and cells were cultured for 3 or 7 days. To measure cell viability, 20 μl of Cell Titer Glo Luminescent cell viability reagent (Promega, G7570) was added into each well, and plates were shaken in the dark for 5 min. Luminescent values were measured by a microplate reader with the Gen5 software (BioTek). Experiments were performed in triplicate.
Kiyokawa J., Kawamura Y., Ghouse S.M., Acar S., Barcin E., Martínez-Quintanilla J., Martuza R.L., Alemany R., Rabkin S.D., Shah K, & Wakimoto H. (2020). Modification of extracellular matrix enhances oncolytic adenovirus immunotherapy in glioblastoma. Clinical cancer research : an official journal of the American Association for Cancer Research, 27(3), 889-902.
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7

Estradiol-Induced Cell Viability Assay

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A total of 2,000 cells were plated in 200 μl medium in clear-bottom black 96-well plates (catalog no. 655090; Greiner Bio One). The following day, the medium was removed from cells and replaced with 200 μl medium containing different concentrations of 17β-estradiol. Cells were then incubated for 48 h. Afterwards, 25 μl of reconstituted CellTiter-Glo luminescent cell viability reagent (catalog no. G7571; Promega) was added to each well and incubated for 5 min. Luminescence readings were taken using the BioTek Synergy H1 hybrid reader. Viability percentages were calculated by normalizing to the readings for dimethyl sulfoxide (DMSO)-treated cells, utilizing wells containing only medium as blanks for a control. DMSO wells were normalized to 100%.
Bristol M.L., James C.D., Wang X., Fontan C.T, & Morgan I.M. (2020). Estrogen Attenuates the Growth of Human Papillomavirus-Positive Epithelial Cells. mSphere, 5(2), e00049-20.
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8

Cell Viability Assay for Trametinib

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Cells (3,000–5,000) were plated per well in 96-well plates 24 h before drug treatment. The number of viable cells was determined 72 h after the initiation of drug treatment using CellTiter-Glo luminescent cell viability reagent according to the manufacturer’s protocol (Promega). Each assay consisted of four replicate wells and was repeated at least three times. Data are expressed as percentage of the cell viability of control cells. Data were graphically displayed using GraphPad Prism version 5.0 for Mac (GraphPad Software). IC50 values were calculated as 50% of growth inhibition as measured by the cell viability assays. Maximal growth inhibition was calculated as the maximum percentage of growth inhibition achieved upon trametinib treatment from 0.1 nM to 1,000 nM, as measured by the cell viability assays.
Lin L., Sabnis A.J., Chan E., Olivas V., Cade L., Pazarentzos E., Asthana S., Neel D., Yan J.J., Lu X., Pham L., Wang M.M., Karachaliou N., Cao M.G., Manzano J.L., Ramirez J.L., Torres J.M., Buttitta F., Rudin C.M., Collisson E.A., Algazi A., Robinson E., Osman I., Muñoz-Couselo E., Cortes J., Frederick D.T., Cooper Z.A., McMahon M., Marchetti A., Rosell R., Flaherty K.T., Wargo J.A, & Bivona T.G. (2015). The Hippo effector YAP promotes resistance to RAF- and MEK-targeted cancer therapies. Nature genetics, 47(3), 250-256.
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9

Lentiviral Transduction and Cytotoxicity Assay

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Ba/F3 cells (4.0 × 105) were infected with lentivirus in the presence of 10 μg/mL polybrene (Sigma-Aldrich) by centrifugation at 3,000 × g for 150 minutes at 32°C. Following overnight incubation at 37°C in 5% CO2, the cells were seeded into 24-well plates and selected in medium containing IL3 and 8 μg/mL blasticidin (Invitrogen) for 10 days. The blasticidin-resistant cells were grown in IL3-free medium for 2 weeks. The expression of exogenous RET proteins was confirmed by immunoblotting. For the drug treatment, 5 × 103 Ba/F3 cells were plated in quadruplicate in 96-well plates and serially diluted inhibitors were added to the wells. Cell viability was measured at 72 hours after drug treatment using the CellTiter-Glo luminescent cell viability reagent (Promega) with EnVision (PerkinElmer). Cell viability was calculated as the cell count in drug-treated samples relative to that in untreated samples (n = 6). The data were displayed graphically using GraphPad Prism version 9.0 (GraphPad Software Inc., RRID:SCR_002798).
Tabata J., Nakaoku T., Araki M., Yoshino R., Kohsaka S., Otsuka A., Ikegami M., Ui A., Kanno S.I., Miyoshi K., Matsumoto S., Sagae Y., Yasui A., Sekijima M., Mano H., Okuno Y., Okamoto A, & Kohno T. (2022). Novel Calcium-Binding Ablating Mutations Induce Constitutive RET Activity and Drive Tumorigenesis. Cancer Research, 82(20), 3751-3762.
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10

Measuring Cellular Metabolic Indicators

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CellTiter-Glo® Luminescent Cell Viability reagent (Promega) was used for ATP measurement. Intracellular glutamate levels were measured using Glutamine/Glutamate-Glo™ Assay kit (Promega). αKG was measured using a colorimetric assay kit (Sigma Aldrich). All kits were used according to manufacturer’s instructions.
Kumar A., Yarosz E.L., Andren A., Zhang L., Lyssiotis C.A, & Chang C.H. (2022). NKT cells adopt a glutamine-addicted phenotype to regulate their homeostasis and function. Cell reports, 41(4), 111516.
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