Alamarblue cell viability reagent

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, Spain, Germany, Canada, Czechia, Poland, Switzerland

AlamarBlue Cell Viability Reagent is a solution used to assess the metabolic activity of cells in culture. It works by measuring the reduction of a redox indicator dye, which changes color and fluorescence in response to chemical reduction of growth medium, reflecting the metabolic activity of cells.

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Lab products found in correlation

430 protocols using alamarblue cell viability reagent

Evaluating Cell Viability in Organoids

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Cell viability was measured using AlamarBlue™ cell viability reagent (DAL1100; Invitrogen) following the manufacturer’s instructions. Briefly, ICOs were distributed equally in 96 well plates (20 μL hydrogel per well) and differentiated to CLCOs for seven days in CDM. CLCOs were treated with different concentrations of CPZ (0–320 μM) with or without BA cocktail in the exposure medium for 24 and 72 h. After exposure, the stock solution of AlamarBlue cell viability reagent was diluted 1:10 in DMEM/F12 without phenol red (11029-021; Gibco) and sterilized with 0.22 μm filter and prewarmed at 37 °C. The culture medium was removed and replaced with prewarmed sterilized AlamarBlue solution. Organoids were incubated at 37 °C for 90 min, the solution was immediately transferred into new 96 well plates for measurement. The fluorescence intensity (wavelength excitation/emission = 545 nm/590 nm) of the reagents was measured with a microplate reader (CLARIOstar Plus; BMG LabTech).

Wang Z., Xing C., van der Laan L.J., Verstegen M.M., Spee B, & Masereeuw R. (2024). Cholangiocyte organoids to study drug-induced injury. Stem Cell Research & Therapy, 15, 78.

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Evaluating Cell Viability in Glioma Models

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GL261 cells were plated at 1 × 10⁴ cells for overnight; 24 hours later, GL261, SJPDGF1, or SF7761 cells (1 × 10⁵) were infected with viruses at different multiplicity of infections (MOIs). At indicated time points, cell viability was assessed by alamarBlue Cell Viability Reagent (ThermoFisher Scientific).
For CD4+ T cell co-culture assays, GL261 cells were seeded in 96-well plates in triplicate and treated with CD4+ T cells at an effector:target ratio of 10:1. Then, 48 h later, wells were washed gently 3x in PBS to remove non-adherent cells. Surviving cells were trypsinized and resuspended in PBS and viable cells were counted (viability was assessed by alamarBlue Cell Viability Reagent [ThermoFisher Scientific]).

Wongthida P., Schuelke M.R., Driscoll C.B., Kottke T., Thompson J.M., Tonne J., Stone C., Huff A.L., Wetmore C., Davies J.A., Parker A.L., Evgin L, & Vile R.G. (2020). Ad-CD40L mobilizes CD4 T cells for the treatment of brainstem tumors. Neuro-Oncology, 22(12), 1757-1770.

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Alamar Blue Cell Viability Assay

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Alamar Blue Cell Viability Reagent was purchased from Life Technologies. (Grand Island, NY) Cells were cultured in 96-well-plate and treated with drugs for 72 hours. The manufacturer's instructions were followed to complete the assay. Drug synergy was determined by the combination index methods using CompuSyn software [23 (link)]. CI values less than 1, equal to 1, and greater than 1 represent synergism, additivity, and antagonism, respectively.

Yamaguchi K., Iglesias-Bartolomé R., Wang Z., Callejas-Valera J.L., Amornphimoltham P., Molinolo A.A., Cohen E.E., Califano J.A., Lippman S.M., Luo J, & Gutkind J.S. (2016). A synthetic-lethality RNAi screen reveals an ERK-mTOR co-targeting pro-apoptotic switch in PIK3CA+ oral cancers. Oncotarget, 7(10), 10696-10709.

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Stress Vulnerability Assays in NPCs

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Stress vulnerability assays were performed as previously described (Silva et al., 2016 (link)) (Figure 7B). NPCs were plated and differentiated in 96-well plate format, for eight weeks. Either QC-01–175, QC-03–075 or vehicle (DMSO) were added directly into the media (100 μL) to a final concentration of 5 μM, and incubated for 8 hr at 37°C. Then, each well was treated with either 10 μM of amyloid-beta(1-42) (Enzo Lifesciences, Farmingdale, NY), or vehicle alone, for an additional 16 hr incubation. At 24 hr, viability was measured with the Alamar Blue Cell viability reagent (Life Technologies), according to manufacturer instructions. Readings were done in the EnVision Multilabel Plate Reader (Perkin Elmer, Waltham, MA).

Silva M.C., Ferguson F.M., Cai Q., Donovan K.A., Nandi G., Patnaik D., Zhang T., Huang H.T., Lucente D.E., Dickerson B.C., Mitchison T.J., Fischer E.S., Gray N.S, & Haggarty S.J. (2019). Targeted degradation of aberrant tau in frontotemporal dementia patient-derived neuronal cell models. eLife, 8, e45457.

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Perfluorocarbon Emulsion Protocol

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1-oleoyl (C_18:1) LPA and fluorescein isothiocyanate–dextran (FITC-dextran; 4 kDa) were purchased from Sigma–Aldrich (Milwaukee, WI, USA). The alamar blue cell viability reagent was purchased from Life Technologies (Carlsbad, CA, USA). Bovine serum albumin (BSA) was purchased from New England Biolabs (Ipswich, MA, USA). Crystal violet (0.1% aqueous) was purchased from Ward’s Science (Rochester, NY, USA). Luria-Bertani (LB) agar powder was purchased from ThermoFisher Scientific (Waltham, MA, USA). Similar to previous work with antibiotic-loaded, water-in-PFC emulsions [24 (link),25 (link)], the PFC used was perfluorocycloether/perfluorooctane (FC-770) purchased from 3 M Inc. (St. Paul, MN, USA) and the fluorosurfactant used (“FSH-PEG”) was perfluoropolyether (Krytox 157FSH oil: 7 kDa, n = 41)-polyethelene glycol (PEG: 1 kDa, m = 22)- Krytox 157FSH (i.e., FSH-PEG-FSH) tri-block copolymers synthesized from Krytox 157FS oil purchased from Dupont (Wilmington, DE, USA) [26 (link)]. See Fig. 1 for the molecular structure of the FSH-PEG copolymer.

Nelson D.L., Zhao Y., Fabiilli M.L, & Cook K.E. (2018). In vitro evaluation of lysophosphatidic acid delivery via reverse perfluorocarbon emulsions to enhance alveolar epithelial repair. Colloids and surfaces. B, Biointerfaces, 169, 411-417.

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Quantifying Titanium Effects on Cellular Viability

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Cellular viability of 100% was attributed to control wells, where cells were cultured with no Ti discs (positive growth control). Cellular viability was quantified via a colorimetric assay using an Invitrogen alamarBlue™ Cell Viability Reagent (DAL1100, lot: 2120063 (Life Technologies Corporation, Thermo Fisher Scientific, Waltham, MA, USA)). Cell viability was measured at days 1, 3, 5, and 10 of cell growth. HOB cells were exposed to alamarBlue™ (1:10, reagent:OGM) for 1 h at 37 °C at each time point. Then, 100 µL of supernatant was transferred into a 96-well plate in triplicates for analysis at each time point. The 96-well plate (Corning Costar Ultra-Low Attachment Multi-Well Plates (Corning Inc., Corning, NY, USA)) was read with a UVM 340 microplate reader at 570 nm and 600 nm (ASYS, Scientific Laboratory Supplies). Cell viability was calculated according to the following equation [32 ]:

Cell viability % = \frac{A 570 - (A 600 x R_{\circ}) for test well}{A 570 - (A 600 x R_{\circ}) positive growth control} \times 100

where A570 and A600 are the absorbance at 570 and 600 nm, respectively, and

R_{\circ}

is the correction factor calculated from A570/A600 for the positive growth control.

Osman M.A., Alamoush R.A., Kushnerev E., Seymour K.G., Shawcross S, & Yates J.M. (2022). In-Vitro Phenotypic Response of Human Osteoblasts to Different Degrees of Titanium Surface Roughness. Dentistry Journal, 10(8), 140.

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Evaluating NPC Viability under Neurotoxic Conditions

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Performed as previously described²¹ and summarized in Supplementary Fig. 4f. NPCs were plated (110,000 cells/cm²) and differentiated in 96-well plate format, for 8 weeks. mTORi or DMSO (Sigma) were added directly onto the media and incubated for 8 h at 37 °C. Then, each well was treated with 30 μM Aβ(1-42), 400 μM NMDA (Supplementary Table 2) or vehicle alone, for an additional 16 h. At 24 h, viability was measured with the Alamar Blue Cell Viability Reagent (Life Technologies) and with the EnVision Multilabel Plate Reader (Perkin Elmer). Calculations and statistics were done in Microsoft Excel version 16.36; graphs were plotted in GraphPad Prism 8 version 8.4.2.

Silva M.C., Nandi G.A., Tentarelli S., Gurrell I.K., Jamier T., Lucente D., Dickerson B.C., Brown D.G., Brandon N.J, & Haggarty S.J. (2020). Prolonged tau clearance and stress vulnerability rescue by pharmacological activation of autophagy in tauopathy neurons. Nature Communications, 11, 3258.

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Cell Viability Assay with Alamar Blue

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Compound or vehicle alone was added directly onto the cellular medium. Viability was measured with the Alamar Blue Cell viability reagent (Life Technologies), according to the manufacturer's instructions. Readings were done in the EnVision Multilabel Plate Reader (PerkinElmer). For IF, fixed neurons image acquisition was done with the IN Cell Analyzer 6000 Cell Imaging System (GE Healthcare Life Sciences). For further details see Supplemental Experimental Procedures.

Silva M.C., Cheng C., Mair W., Almeida S., Fong H., Biswas M.H., Zhang Z., Huang Y., Temple S., Coppola G., Geschwind D.H., Karydas A., Miller B.L., Kosik K.S., Gao F.B., Steen J.A, & Haggarty S.J. (2016). Human iPSC-Derived Neuronal Model of Tau-A152T Frontotemporal Dementia Reveals Tau-Mediated Mechanisms of Neuronal Vulnerability. Stem Cell Reports, 7(3), 325-340.

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Viability Assay for Neuronal Cells

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Assay for neuronal cells cultured in black 96-well plates with a clear bottom (Fisher Scientific Corning), as previously described²¹. Viability was measured with the Alamar Blue Cell viability reagent (Life Technologies) at 1:10 dilution, after 4 h incubation at 37 °C, according to manufacturer’s instructions. Readings were done in the EnVision Multilabel Plate Reader (Perkin Elmer). Calculations were done using Microsoft Excel version 16.36 and graphs were plotted in GraphPad Prism 8 version 8.4.2.

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Establishment of MPNST Cell Lines

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MPNST724 and ST88-14 human MPNST cell lines were obtained from Jonathan A. Fletcher laboratory at DFCI and have been tested mycoplasma free. MPNST 724 was grown in RPMI with 10% FBS and ST88-14 was grown in RPMI with 15% FBS. cDNA for wild-type human EED and SUZ12 in pDONR vectors were obtained from Harvard PlasmidID and cloned into MSCV-based retroviral vector with FLAG-HA (FH) tag (Addgene plasmid 41033)^{43 (link)} using Gateway technology. To generate stably expressing cell lines, MPNST724 and ST88-14 were infected with empty vector, MSCV-FH-EED, MSCV-FH-SUZ12 and selected using puromycin (2 μg/ml for 72 hours). Growth curve of the infected cells was performed using Alamar blue cell viability reagent (Life Technology, DAL1100).
For immunofluorescence of infected cell lines, cells were fixed in 4% paraformaldehyde for 10 minutes, permeabilized in 0.1% Triton X-100, and blocked for 1 hours using 10% goat serum. The cells were then incubated for 2 hours in primary antibody (H3K27me3, #9733, Cell Signaling, 1:400) followed by secondary antibody (Alexa-594 conjugated goat-anti-rabbit, Invitrogen). Slides were mounted using Prolong Gold with DAPI (Invitrogen). Photographs were taken on a Nikon microscopy using a Roeper Scientific camera.

Lee W., Teckie S., Wiesner T., Ran L., Prieto Granada C.N., Lin M., Zhu S., Cao Z., Liang Y., Sboner A., Tap W.D., Fletcher J.A., Huberman K.H., Qin L.X., Viale A., Singer S., Zheng D., Berger M.F., Chen Y., Antonescu C.R, & Chi P. (2014). PRC2 is recurrently inactivated through EED or SUZ12 loss in malignant peripheral nerve sheath tumors. Nature genetics, 46(11), 1227-1232.

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