Live dead viability cytotoxicity kit

Manufactured by Merck Group

Sourced in United States

The Live/Dead Viability/Cytotoxicity kit is a lab equipment product that provides a quantitative assay to distinguish live from dead cells based on membrane integrity. The kit uses two fluorescent dyes to label cells, with one dye staining only dead cells and the other staining all cells. The resulting fluorescence is measured to determine the ratio of live to dead cells in a sample.

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5 protocols using live dead viability cytotoxicity kit

Evaluating Cell Viability in μG Experiments

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To verify both the biocompatibility of MOC platform and assess viability of Human Glioblastoma A-172 and HUVEC cells after μG experiments, live imaging using LIVE/DEAD Viability/Cytotoxicity Kit (Sigma-Aldrich, Cat. No. L3224) was performed as per manufactures instructions. Briefly, fluorescent calcein-AM (2 μM) and red-fluorescent ethidium homodimer-1 (EthD-1) (4 μM) were gently injected into the MOC and incubated for 15 min at 37 °C and humidify atmosphere. MOCs were then washed with PBS and imaged using EVOS M5000 microscope (Invitrogen). To evaluate proliferation, cells were detached from the MOC using Trypsin-EDTA, collected, centrifugated and counted using a Hemocytometer. Trypan Blue 0.4% (Thermofisher Scientific, Cat. No. 15250061) was used to determine the percentage of viable cells present in the suspension.

Silvani G., Bradbury P., Basirun C., Mehner C., Zalli D., Poole K, & Chou J. (2022). Testing 3D printed biological platform for advancing simulated microgravity and space mechanobiology research. NPJ Microgravity, 8, 19.

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Chondroitin Sulfate A Hydrogel Encapsulation

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Chondroitin sulfate A, methacrylic anhydride and NIPAAm monomer were all purchased from Sigma-Aldrich. NIPAAm was purified in excess n-hexane and recrystallized prior to use. Lipids 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-glycero-3-phosphocholine (DPPC) were purchased from Avanti Polar Lipids. Gelatin was purchased from MP Biomedical, with an average molecular weight ranging from 20 kDa to 100 kDa. High-glucose (4.5g/L) Dulbecco’s Modified Eagle’s Medium (DMEM), Dulbecco’s Phosphate-Buffered Saline (DPBS), heat inactivated fetal bovine serum (FBS), trypsin and penicillin-streptomycin (Pen-Strep) for cell cultures were purchased from Life Technologies. DNeasy Blood and Tissue kit for extraction of cellular DNA was purchased from Qiagen. PicoGreen dsDNA Assay kit for cellular DNA quantification was purchased from Life Technologies. In Vitro Toxicology Assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT)- based, and Live/Dead Viability/Cytotoxicity kit, for mammalian cells, were purchased from Sigma-Aldrich and Life Technologies, respectively, to study the viability of polymer-encapsulated cells. All solvents were of analytical grade. Fresh porcine cartilage, from pig ears, was obtained from a butcher.

Wiltsey C., Christiani T., Williams J., Scaramazza J., Van Sciver C., Toomer K., Sheehan J., Branda A., Nitzl A., England E., Kadlowec J., Iftode C, & Vernengo J. (2015). Thermogelling Bioadhesive Scaffolds for Intervertebral Disc Tissue Engineering: Preliminary In Vitro Comparison of Aldehyde-Based Versus Alginate Microparticle-Mediated Adhesion. Acta biomaterialia, 16, 71-80.

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Fabrication of Photoactive Polymer Hydrogels

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2,4,6-trimethyl benzoyl diphenyl phosphine oxide (TPO), Gelatin, Methacrylic anhydride (MAA), Poly(ethylene glycol) diacrylate (PEGDA, Mn 700), N-butyl acetate (nBuAc), 2-propanol (IPA), sodium dodecyl sulfate (SDS), polyvinylpyrrolidone (PVP), Polydimethylsiloxane (PDMS), sodium carbonated, sodium bicarbonate, sodium hydroxide, hydrochloric acid, DAPI and Live/Dead viability/cytotoxicity kit including PI and annexin were purchased from Sigma Aldrich. Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), pen-strep, and trypsin were purchased from Gibco. Collagenase was obtained from Merck. All of the reagents were used without any purification.

Ghazali H.S., Askari E., Seyfoori A, & Naghib S.M. (2023). A high-absorbance water-soluble photoinitiator nanoparticle for hydrogel 3D printing: synthesis, characterization and in vitro cytotoxicity study. Scientific Reports, 13, 8577.

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Viability of DPSCs in 3D Cultures

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Viability of DPSCs in 3-dimensional cultures of GM and FM was assessed over 15 days using MTT assay. Briefly, after incubation of the scaffolds with 0.5 mg/mL of methylthiazolyldiphenyl-tetrazolium bromide (MTT, Sigma-Aldrich) for 60 min at 37 °C with 5% CO2, the converted dye was eluted with DMSO for 3 h. Absorbance was measured at λ = 570 nm. For each time point, measurement of cells without scaffolds served as a reference and was set to 100%.
To visualize live and dead cells, live/dead staining was performed on the 5th, 10th, and 15th days of in vitro cell culturing. A Live/Dead Viability/Cytotoxicity kit (Sigma-Aldrich) was used per the manufacturer’s instructions with slight modifications. Briefly, 10 μL of calcein-AM and 5 μL of propidium iodide solution were added to 5 mL of PBS to prepare the assay solution. The prepared scaffold containing the cells was washed with PBS 10 times to remove residual esterase activity, and 200 μL of assay solution was added to the scaffold and incubated at 37 °C for 30 min. Fluorescence was detected using an inverted fluorescence microscope (DS-Ri2, Nikon, Tokyo, Japan).

Jang J.H., Moon J.H., Kim S.G, & Kim S.Y. (2020). Pulp regeneration with hemostatic matrices as a scaffold in an immature tooth minipig model. Scientific Reports, 10, 12536.

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Antimicrobial Silver Nanoparticle Synthesis

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Silver nitrate, Potassium tetrachloropalatinate (II), Ascorbic acid, Sodium chloride, Brij58, Tryptic soy broth (TSB), Fetal bovine serum (FBS), Penicillin-streptomycin solution, Trypsin and LIVE/DEAD™ Viability/Cytotoxicity Kit, were purchased from Sigma-Aldrich, United States. Dulbecco’s Modified Eagle’s Medium (DMEM) and Fluorescent solution BacLight™ bacterial viability kit, were obtained from Thermo Fisher, United States. CellTiter 96^® AQueous One Solution Cell Proliferation assays (MTS) was acquired from Promega, United States. All chemicals were used as received.

López Ruiz A., Bartomeu Garcia C., Navarro Gallón S, & Webster T.J. (2020). Novel Silver-Platinum Nanoparticles for Anticancer and Antimicrobial Applications. International Journal of Nanomedicine, 15, 169-179.

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