Viability of cortical cells and microglia were measured by metabolism of thiazolyl blue, 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma Aldrich) and CellTitre-Glo®(Promega, Thermo Fisher Scientific, Waltham, MA). For MTT, injured and uninjured cortical cells cultured with and without microglia were incubated with 100 µl MTT in 1 ml of media for 1 h. Media was removed and cells were dissolved in 300 µl dimethylsulfoxide (DMSO) and aliquoted to 100 µl/well in 96-well plates. Absorbance was read at 540-590 nm on an ELISA plate reader. Three experiments were performed in triplicate. For CellTitre-Glo® manufacturer’s protocol was followed (https://www.promega.com/-/media/files/resources/protocols/technical-bulletins/0/celltiter-glo-luminescent-cell-viability-assay-protocol.pdf?la=en ). Briefly, injured and uninjured cortical cells cultured with and without microglia were incubated with pre-mixed CellTitre-Glo®, Reagent equal to the cell culture medium volume, cells and Reagent were misted for 2 min to allow for cellular lysis, and the plate was incubated for 10 min. For each condition triplicate 100 µl samples were placed into 96 well plates and chemiluminescence was recorded to measure ATP and metabolic activity of viable cells (BioTek Synergy, Winooski, VT, USA).
Celltitre glo
Manufactured by Promega
Sourced in United States, United Kingdom
CellTiter-Glo is a reagent used to measure the number of viable cells in a sample. It quantifies the amount of ATP present, which is an indicator of metabolically active cells. The assay is based on the luciferase reaction, which produces luminescence that can be detected and correlated to the number of living cells.
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Lab products found in correlation
Foetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Wst 1, by Roche (2 mentions)
Alamarblue assay, by Thermo Fisher Scientific (2 mentions)
Pci vsvg, by Addgene (2 mentions)
Lentiviral shrna clones, by Merck Group (2 mentions)
Shc007, by Merck Group (2 mentions)
Pspax2, by Addgene (2 mentions)
Shc002, by Merck Group (2 mentions)
Mycoalert detection kit, by Lonza (2 mentions)
Sheep blood agar plates, by Hardy Diagnostics (2 mentions)
Amaxa nucleofection kit, by Lonza (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Interleukin 6 il 6, by Miltenyi Biotec (2 mentions)
Fluostar optima, by BMG Labtech (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Cisplatin, by Spectrum Chemical (1 mentions)
Non adherent 96 well plate, by Sarstedt (1 mentions)
67 protocols using celltitre glo
1
Cortical Cell and Microglia Viability Assay
2
Murine Tumor Dissociation and Sphere Formation
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Upon murine necropsy, Total tumor collected per mouse was dissociated using standard methods with a Papain dissociation kit (Worthington Biologicals). Following ltration through a 40 micron lter, single cells were counted cultured in serial dilutions in a non-adherent 96-well plate (Sarstedt) with 200 μl serum-free DMEM/F12 medium supplemented with 20 ng/mL basic broblast growth factor (Invitrogen), 10 ng/mL epidermal growth factor (BioSource), and 2% B27 (vol/vol) (Invitrogen). Tumorsphere-formation was scored following 2 weeks incubation using a phase contrast microscope. The sphere initiation frequency was calculated using an extreme limiting dilution algorithm (ELDA) (http://bioinf.wehi.edu.au/software/elda/). In vitro ABX cisplatin sensitivity studies ID8-LUC and ID8-VEGF cells were plated at 1,000 cells per well and treated with ampicillin, vancomycin, metrinodazole, and neomycin in combinations of the same ratios used in the murine studies for 72 hours, wherein proliferation was measured using Cell Titre Glo (Promega) and compared to vehicle treated control. Additionally following incubation for 7 days with ampicillin, vancomycin, metrinodazole, and neomycin the IC50 of cisplatin (Spectrum Chemical) was assessed compared to vehicle pretreated controls also utilizing Cell Titre Glo (Promega).
3
α-Synuclein Transfection and Cell Viability
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SH-SY5Y cells were seeded onto tissue culture treated 96-well plate and then transfected with EGFP-α-synuclein only, and/or co-transfected with siRNA. To cells, appropriate drugs were added (24 h) after 48 h of transfection. Using luminescence-based CellTitre-Glo® (Promega) kit, the cell viability was assayed using automated microtitre plate reader Varioskan Flash (Thermo Scientific).
4
Evaluating BRCA2 and CBLC Knockdown Effects
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Cell lines were transfected with SMARTpool siRNAs (Dharmacon, GE Healthcare) targeting BRCA2 and CBLC or siCONTROL A (Santa Cruz Biotech) using RNAiMax (Invitrogen) transfection reagent. Cell lines were infected with GIPZ shRNA constructs packaged as lentivirus and after 72hrs selected with 2μg/ml puromycin.
Clonogenic survival assays were performed as previously described [3 (link)]. Short-term survival assays were performed in 96-well plates. Cells were seeded in 96-well plates and drug was added after 24 hours. Cell viability was estimated after seven days using Cell-Titre Glo (Promega). Surviving fractions (SFs) were calculated and drug sensitivity curves plotted as previously described [3 (link)].
Clonogenic survival assays were performed as previously described [3 (link)]. Short-term survival assays were performed in 96-well plates. Cells were seeded in 96-well plates and drug was added after 24 hours. Cell viability was estimated after seven days using Cell-Titre Glo (Promega). Surviving fractions (SFs) were calculated and drug sensitivity curves plotted as previously described [3 (link)].
5
Cytotoxicity and Apoptosis Assay of Cancer Drugs
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Cell lines were plated in quintuplicate in 96-well flat-bottom plates with idelalisib, CZC24832 and duvelisib. These were incubated for 24–72 h with viable numbers being measured using Cell Titre GLO (Promega, Southampton, UK). Flow cytometry was performed to measure apoptosis using the CyFlow Cube 6 flow cytomter (Sysmex, Milton Keynes, UK). For measuring viability, samples were collected and stained with Annexin V and propidium iodide (PI), followed by detection via flow cytometry. Data were then normalised to vehicle controls. All data points are represented as the mean with s.d.
6
Evaluating Antibody-Drug Conjugate Efficacy
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Example 9
This example provides the results of EC50 assays (nM) of the designated drug conjugated antibodies measured in vitro in specified cells. The antibody used was an anti-HER2 IgG class of antibody. Seven breast cancer cell lines with various level of Her2 expression as indicated with plus or minus signs in the table below were plated in 96 well plate. The ADCs were serial diluted and added onto cells for treatment for 5 days. At the end of the study, cell proliferation was measured by Promega's CellTitreGlo. EC50 (in nM) was determined as the concentration of 50% cell growth inhibition. The selection criteria for a successful compound included high efficacy, such as killing cell lines with high expression of the target receptor, with EC50 less than 3 nM. Also, the successful candidate should have low toxicity and good therapeutic window, as determined by relatively low killing of the control cell line (MDA468) with low expression of the target receptor. Both ADCs 22 (
7
Quantifying Cell Viability via ATP
8
Phytochemical Cytotoxicity Evaluation in Breast Cancer
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Breast cancer cells (MCF7, T47D, MDA-MB-231, and BT-549) were seeded in 12-well, 24-well or 96-well plates at 10%~20% confluency in a medium with 10% FBS and 1% penicillin/streptomycin. After overnight incubation, cells were treated with different concentrations of phytochemicals (1–512 µM, 2-fold dilution series) and returned to the CO2 incubator for the measurement of cell viability at different time points (24, 48, and 72 h) using a metabolic assay (CellTitre-Glo, Promega or WST-1, Roche, Basel, Switzerland). Data are analyzed to generate a dose-response curve (nonlinear curve fitting) and growth rate inhibition (GRI). The concentrations at 20% (EC20) or 50% (EC50) response were determined by OriginPro.
9
Compound Screening for Cell Viability Assay
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Cells (2000/well) were seeded in a 96-well plate and treated with inhibitors at the indicated doses for 72 h prior to assessment of cell viability using Cell Titre Glo (Promega), following the manufacturer's recommendations. IC50 data were generated from dose-response curves fitted using a four-parameter regression fit in GraphPad Prism 6 software. Inhibitors used in this study include Gefitinib, Rociletinib, Lapatinib, Neratinib, Sorafenib, Ceritinib, Crizotinib, Pazopanib, Sunitinib, Dasatinib, Ponatinib, AZD4547, Bosutinib, BEZ235, Trametinib, NVP-AUY-922, Imatinib (LC laboratories) AZD9291, PF-562271, Palbociclib, BGJ398, MK2206, AZD5363 (Selleck Chemicals), BX-795, MRT67307 (Sigma-Aldrich), JQ1 (Cayman Chemical Company), DDR1-in-1 (Tocris), CCT244747 (ICR).
10
Antibody-Mediated Complement Cytotoxicity
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Tumor cells were resuspended in serum-free media and seeded in 96-well white flat bottom plates at 5 × 104 cells/well. Cells were incubated with pooled NHS for 5 min at 37 °C, 5% CO2 in the presence or absence of 0.01 M EDTA. Serial dilutions of antibody were added to the wells such that the final concentration of NHS was 25%. Plates were incubated for 2.5 h at 37 °C, 5% CO2. Cell viability was assessed by Cell Titre-Glo® (Promega) luminescence using the BioTek Synergy plate reader.
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