Alamar blue

Manufactured by Tecan

Sourced in Switzerland, United States

Alamar Blue is a cell-based assay reagent used to assess cell viability and proliferation. It measures the redox potential of cells, which changes as cells grow, divide, and respond to various stimuli. Alamar Blue is a non-toxic, water-soluble dye that can be added directly to cell cultures, with no sample preparation required.

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Lab products found in correlation

Alamar blue, by Thermo Fisher Scientific (24 mentions) Infinite m200 pro, by Tecan (3 mentions) F200 spectrophotometer, by Tecan (3 mentions) Infinite 200 pro, by Tecan (3 mentions) Alamarblue reagent, by Thermo Fisher Scientific (2 mentions) Ab126572, by Abcam (2 mentions) Infinite f200 pro, by Tecan (2 mentions) Crystal violet, by Merck Group (2 mentions) Reagent diluent, by R&D Systems (1 mentions) 96 well cell culture plate, by Corning (1 mentions) Infinite 4300, by Tecan (1 mentions) Phosphatase inhibitor cocktail 2, by Merck Group (1 mentions) Infinite 200, by Tecan (1 mentions) Phosphate buffered saline solution, by Thermo Fisher Scientific (1 mentions) M1000 pro, by Tecan (1 mentions) Alamar blue, by Merck Group (1 mentions) Human umbilical vein endothelial cells (huvec), by Lonza (1 mentions) Egm 2mv, by Lonza (1 mentions) Infinite m200 pro spectrophotometer, by Tecan (1 mentions) Spectrafluor plus, by Tecan (1 mentions)

29 protocols using alamar blue

Metabolic Activity of Osteoblast-like Cells

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To assess the presence of metabolically active MG-63 and Saos-2 cells seeded onto GEN/EtOH or GEN/PBS crosslinked samples, the one-step Alamar Blue assay (Invitrogen, Carlsbad, CA, USA, DAL1100) was performed according to the manufacturer’s instructions. Briefly, the culture medium was removed, replaced with the Alamar Blue solution prepared as 10% v/v in fresh cell culture medium and incubated at 37 °C, 95% humidity for 4 h. Then the fluorescence of Alamar Blue solution was quantified by using a microplate reader (Infinite F200 PRO, TECAN, Mannedorf, Switzerland) at 535 nm excitation and 590 nm emission wavelengths. Data are expressed as a percentage of control cultures (i.e., MG-63 and Saos-2 seeded in tissue culture plates) and reported as mean value ± standard deviation of triplicates. The hybrid material without cells was also analyzed and considered as background.

Montalbano G., Borciani G., Cerqueni G., Licini C., Banche-Niclot F., Janner D., Sola S., Fiorilli S., Mattioli-Belmonte M., Ciapetti G, & Vitale-Brovarone C. (2020). Collagen Hybrid Formulations for the 3D Printing of Nanostructured Bone Scaffolds: An Optimized Genipin-Crosslinking Strategy. Nanomaterials, 10(9), 1681.

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Quantifying HA Secretion in 4T1 Cells

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4T1 murine breast carcinoma cells were seeded in 96 well cell culture plates (Corning Costar) at 5,000 cells per well. Cells were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum. MU was solubilized in dimethyl sulfoxide (DMSO), diluted into RPMI 1640 media supplemented with insulin, transferrin, and sodium selenite (ITS, Sigma)), and added to cells. MU-P was solubilized directly into RPMI-ITS and added to cells. Twenty-four hours after plating, cells were washed 3x with phosphate buffered saline (2.7mM KCl, 1.5mM KH₂PO₄, 136.9mM NaCl, 8.9mM Na₂HPO₄, pH 7.4; PBS). MU and MU-P were added to cells after washing with or without Phosphatase Inhibitor Cocktail 2 (Sigma). Forty-eight hours later, 50 μL of media was removed and frozen. Fifteen μL of alamarBlue (Life Technologies) was added to cell media and incubated for 3 hours. Cell number was quantified by measuring alamarBlue fluorescence at 585 nm after excitation at 570 nm using a Tecan Infinite 4300 (Tecan Group Ltd).
HA levels in culture media were quantified by using the HA DuoSet ELISA assay (R&D Systems). Frozen media samples were thawed and diluted 1:100 in Reagent Diluent (R&D Systems). The ELISA was run according to the manufacturer’s specifications. Total HA in culture media was normalized to cell number using the alamarBlue assay and to DMSO control wells.

Kohli A.G., Kivimäe S., Tiffany M.R, & Szoka F.C. (2014). Improving the distribution of Doxil® in the tumor matrix by depletion of tumor hyaluronan. Journal of controlled release : official journal of the Controlled Release Society, 191, 105-114.

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Cell Viability Assay in 96-well Plate

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Cells were seeded in a 96-well plate at a concentration of 1x10⁴ cells/mL and treated 16 hr after seeding. Cell viability was determined using the AlamarBlue^® (Invitrogen), 24 hr after treatment. Fluorescence of the AlamarBlue^® reagent was read using a peak excitation at 570 nm and peak emission at 585 nm using a Tecan Infinite M1000 scanner (Tecan Group Ltd).

Uribe D.J., Mandell E.K., Watson A., Martinez J.D., Leighton J.A., Ghosh S, & Rothlin C.V. (2017). The receptor tyrosine kinase AXL promotes migration and invasion in colorectal cancer. PLoS ONE, 12(7), e0179979.

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Cytotoxicity of 5-FC in AMLV-CD and GALV-CD Cells

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Cells at 20–30% confluence were infected with AMLV–CD or GALV–CD at an MOI of 0.01. On day 15, 5-FC (Tokyo Chemical Industry, Tokyo, Japan) was added to the culture medium at final concentrations of 0.1, 1, or 10 mM. On day 21, the percentage of viable cells was determined with alamarBlue (Thermo Fisher Scientific Inc., Waltham, MA, USA), in accordance with the manufacturer’s instructions. Briefly, 10% alamarBlue reagent was added aseptically to the culture medium, which was then incubated for 3 h. Fluorescence was measured with an Infinite M200 PRO microplate reader (Tecan Group Ltd., Männedorf, Zurich, Switzerland) at an excitation wavelength of 544 nm and a fluorescence wavelength of 590 nm. The percentage of viable cells was determined by calculating the fluorescence of viable cells measured against wells without 5-FC.

Sonoda-Fukuda E., Takeuchi Y., Ogawa N., Noguchi S., Takarada T., Kasahara N, & Kubo S. (2024). Targeted Suicide Gene Therapy with Retroviral Replicating Vectors for Experimental Canine Cancers. International Journal of Molecular Sciences, 25(5), 2657.

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Cell Viability Assay for Biomaterials

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AlamarBlue (Invitrogen, Carlsbad, CA, USA) was used to measure the cell viability after 72 h of incubation with DCPD, OCP, and HAp. The cells were incubated with 100 μg/mL AlamarBlue for 4 h at 37 °C in a moist environment of 5% CO₂, and the mean fluorescence intensity (MFI) of the resultant resofurin product was measured using an Infinite^®200 microplate reader (Tecan Group Ltd., Männedorf, Switzerland): Ex530 nm/Em 595 nm. The vitality of the control cells that had not been treated with DCPD, OCP, or HAp was assumed to be 100%. Cell viability was measured as a percentage relative to the control using the following formula: Cell viability (%) = (MFI cells after incubation with DCPD, OCP, and HAp/MFI of control cells) × 100% [19 (link),33 (link)].

Minaychev V.V., Smirnova P.V., Kobyakova M.I., Teterina A.Y., Smirnov I.V., Skirda V.D., Alexandrov A.S., Gafurov M.R., Shlykov M.A., Pyatina K.V., Senotov A.S., Salynkin P.S., Fadeev R.S., Komlev V.S, & Fadeeva I.S. (2024). Low-Temperature Calcium Phosphate Ceramics Can Modulate Monocytes and Macrophages Inflammatory Response In Vitro. Biomedicines, 12(2), 263.

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Alamar Blue Viability Assay

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At day 5 (D5, occurring just prior to the inoculation of the bacteria) and day 7 (D7, occurring at the end of culture), the culture supernatant was removed and replaced with culture medium containing 10% Alamar Blue (Fisher Scientific, Illkirch, France #DAL1025). After incubation for 1 h at 37 °C, the medium containing Alamar Blue was collected, and the cells were rinsed with Phosphate Buffered Saline solution (Fisher Scientific) before being returned to the culture medium and inoculated (D5) or fixed for further analysis (D7).
For each condition, the fluorescence intensity (FI) emitted by the solution containing Alamar Blue was measured using a fluorometer TECAN Spark^®, Lyon, France) at a rate of 3 measurements per well and using the following wavelengths: excitation at 570 nm and emission at 585 nm.

Forraz N., Bize C., Desroches A.L., Milet C., Payen P., Chanut P., Kern C., Garcia C, & McGuckin C. (2023). The World’s First Acne Dysbiosis-like Model of Human 3D Ex Vivo Sebaceous Gland Colonized with Cutibacterium acnes and Staphylococcus epidermidis. Microorganisms, 11(9), 2183.

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Evaluating Metabolic Activity of pASC on Mineralized Scaffolds

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Metabolic activity of pASC seeded on mineralized collagen and mineralized collagen–amnion scaffolds was calculated using a non-destructive alamarBlue^® assay over the course of 28 days (Days 0, 4, 7, 14 and 28). Six scaffolds were rinsed in PBS prior to incubation under gentle shaking in alamarBlue^® (Invitrogen, Carlsbad, CA, USA) in an incubator at 37°C for 2 h. Following incubation, the alamarBlue^® solution was measured using a F200 spectrophotometer (Tecan, Mannedorf, Switzerland) for the fluorescence of resorufin (540(52)-nm excitation, 580(20)-nm emission). Scaffold metabolic activity at Days 0 through 28 was calculated from a standard curve generated on Day (−1) of known numbers of cells and normalized to the cell seeding density of 100 000 cells.

Dewey M.J., Johnson E.M., Slater S.T., Milner D.J., Wheeler M.B, & Harley B.A. (2020). Mineralized collagen scaffolds fabricated with amniotic membrane matrix increase osteogenesis under inflammatory conditions. Regenerative Biomaterials, 7(3), 247-258.

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Cytotoxicity Assay of Macrophages

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BALB/c mice were killed by CO₂ inhalation and their peritoneal macrophages were collected by washing the abdominal cavity with cold RPMI-1640 culture medium supplemented with antibiotics (sodium penicillin 200 UI and streptomycin 200 µg/mL) and 10% fetal bovine serum. The macrophages were distributed in 96-well culture plates at 10⁵ cells/well. After 4 h incubation at 37 °C and 5% CO₂, the culture medium was replaced by fresh medium containing test compounds and the plates were incubated for other 48 h. Afterwards, 10 µL Alamar Blue (DAL1025, Thermo Fisher Scientific) was added per well. After other 6–8 h of incubation (37 °C and 5% CO₂), the reduction of Alamar Blue by viable cells was assessed by reading fluorescence at EX/EM 530/585 nm (cutoff, 550 nm) in a Tecan Infinite 200 Pro microplate reader. Fifty percent cytotoxic concentrations (CC₅₀) were estimated by non-linear fitting to the Emax sigmoid model [31 (link)].

Sifontes-Rodríguez S., Mollineda-Diogo N., Monzote-Fidalgo L., Escalona-Montaño A.R., Escario García-Trevijano J.A., Aguirre-García M.M, & Meneses-Marcel A. (2023). In Vitro and In Vivo Antileishmanial Activity of Thioridazine. Acta Parasitologica, 69(1), 324-331.

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Collagen Cell Viability Assay

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Cell were seeded into collagen matrix (40,000 cells/100 μL of collagen matrix; 4 technical replicates per sample) to a 96-well plate, overlaid by DMEM without phenol red containing 1% FBS (100 μL) and cultivated for 48 h. Collagen without cells served as a blank for the experiment. After 48 h, overlaying medium was replaced by medium containing AlamarBlue reagent (Invitrogen, Carlsbad, CA, USA) in 5:1 ratio and cultivated for another 4 h. Finally, the medium containing AlamarBlue was transferred to new wells and the fluorescence (excitation 550 nm, emission 590 nm) was measured using the Infinite M200 Pro plate fluorimeter (TECAN, Mannedorf, Switzerland). At least three independent experiments were performed and at least 3 technical replicates were analyzed per sample. The data were statistically analyzed in GraphPad Prism 6 using two-way ANOVA.

Merta L., Gandalovičová A., Čermák V., Dibus M., Gutschner T., Diederichs S., Rösel D, & Brábek J. (2020). Increased Level of Long Non-Coding RNA MALAT1 Is a Common Feature of Amoeboid Invasion. Cancers, 12(5), 1136.

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Quantifying Metabolic Activity of Scaffold Variants

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An alamarBlue® assay was used to quantify the metabolic activity of all scaffold variants.²⁷ A standard curve was generated with known cell numbers to correlate fluorescent readings with fold change of metabolic activity. In reported data, a metabolic activity of 1 represents the metabolic activity of the initial scaffold cell seeding density (150 000 cells). Scaffolds were washed in PBS at every timepoint (day 1, 4, 7, 14, 28) and placed in a solution of alamarBlue® (Invitrogen, California, USA) in an incubator at 37 °C on a shaker. After incubation for 1.5 hours, the alamarBlue® solution was collected and fluorescence of resorufin (540(52) nm excitation, 580(20) nm emission) was measured with a F200 spectrophotometer (Tecan, Switzerland) (n = 6).

Dewey M.J., Nosatov A.V., Subedi K, & Harley B. (2020). Anisotropic mineralized collagen scaffolds accelerate osteogenic response in a glycosaminoglycan-dependent fashion. RSC Advances, 10(26), 15629-15641.

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