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The LIVE/DEAD Cell Imaging Kit is a sensitive two-color fluorescence cell viability assay optimized for FITC and Texas Red filters. Calcein AM Calcein AMCalcein494nm517nmPI-DNA535nm617nm The kit identifies live versus dead cells on the basis of membrane integrity and esterase activity. 2B), cell death was quantified by trypan blue exclusion assay. 5 a). Living/dead staining and apoptosis assay. EarlyTox Live/Dead Assay Kit. An ideal cell viability assay should be safe, rapid, reliable, efficient, and time- and cost-effective, and should not interfere with the test compound. AAT Bioquest Live or Dead Cell Viability Assay Kits are based on the simultaneous determination of live and dead cells using two fluorescent probes. Live cells are identified using either a calcein acetoxymethyl (AM) derivative or a mitochondrial membrane potential probe, while dead cells are labeled using a cell-impermeable DNA binding dye. Application: Cell viability analysis using Calcein AM, Propidium Iodide and Hoechst on A549 cells. Next, cells were stained with Calcein-AM/PI dye for 25 min and observed under a fluorescent microscope (Olympus IX71, Japan) at excitation/emission wavelengths of 530/580 nm and 490/515 nm for PI and Calcein-AM, respectively. Wear safety glasses at all times. Features: Form: Lyophilized powder. Add 200 l (for 96 well plate) or 400 l (for a 24 well plate) per well of 3 M calcein AM (live dye) or 2.5-5.0 M PI (dead dye) diluted in warm (37 C) 1X DPBS. The Live Dead assay staining solution is a mixture of two fluorescent dyes that differentially label live and dead cells. It is important that cells be distributed evenly on the bottom of the well, since the The application of chemodynamic therapy (CDT) for cancer is a serious challenge owing to the low efficiency of the Fenton catalyst and insufficient H2O2 expression in cells. Cells were treated with calcein AM dye and ethidium homodimer-1 (Invitrogen) at a final concentration of 1.33 M calcein AM and 2.5 M ethidium homodimer-1. (D) Livedead staining of HeLa cells subjected to the various treatments. Thermo Fisher calcein am ethidium homodimer live dead assay kit Calcein Am Ethidium Homodimer Live Dead Assay Kit, supplied by Thermo Fisher, used in various techniques. Molecular weight: 994.86. Specifically, 4T1 cells were inoculated in a 12-well plate (1 10 5 cells per well). The large quantum yield of Calcein dyes enables them to be readily Live cells and tissues are imaged over long time periods using a fast, non-toxic click chemistry. The live cells were stained with 2 M calcein-AM for 20 min, while the dead cells were stained with 4.5 M propidium iodide (PI) for 20 min. MG-63 cells were plated onto the prepared cement discs for both cements at a density of 1 10 4 cells/mL in 2 mL of culture medium. Next, cells were stained with Calcein-AM/PI dye for 25 min and observed under a fluorescent microscope (Olympus IX71, Japan) at excitation/emission wavelengths of 530/580 nm and 490/515 nm for PI and Calcein-AM, respectively. Live/Dead PTX PTX I am doing a Live/Dead assay using Calcein, AM, for live cells and ethidium homodimer-1 for dead cells. BMM live/dead assays. The cell viability was determined by a LIVE/DEAD viability/cytotoxicity kit for mammalian cells (Thermo Fisher Scientific) by adding 4 M calcein and ethidium homodimer-1 into the culture media. B ROS changed in MCF-7 cells after hydrogen treatment. The Live Dead assay staining solution is a mixture of two fluorescent dyes that differentially label live and dead cells. Quick and easy to use, the kit allows discrimination between live and dead cells with two probes that measure recognized parameters of cytotoxicity and cell viabilityintracellular esterase activity and plasma membrane integrity. Accidental release measures Stop leak if without risk. 8. The LIVE/DEAD Cell Imaging Kit is a sensitive two-color fluorescence cell viability assay optimized for FITC and Texas Red filters. 2011 May; 6(5):513-8. This kit contains calcein-AM and propidium iodide (PI) solutions, which stain viable and dead cells, respectively. Scale bar, 400 m. Similarly, calcein-AM is taken up by live cells where cytoplasmic esterase activity removes the AM group to generate fluorescent calcein which is retained in live cells. MDA MB 231 cells were simultaneously stained with calcein and ethidium using a live-dead assay kit. In live cells the nonfluorescent calcein AM is converted to a green-fluorescent calcein after acetoxymethyl ester hydrolysis by intracellular esterases. To determine the cell viability, BMMs were incubated with spiky particles or rough particles at a dose of 0.005, 0.01, 0.02, 0.04 or (green: living cells; red: dead cells). The fluorescent calcein molecule is restored, which is trapped in the cell and emits strong green fluorescence. The kit identifies live versus dead cells on the basis of membrane integrity and esterase activity. C Fluorescence quantification of ROS. Herein, we fabricated a PDGFB targeting, biodegradable FePt alloy assembly for magnetic resonance imaging (MRI)-guided chemotherapy and starving-enhanced chemodynamic therapy for cancer Cells were resuspended in 300 l of calcein AM (0.2 M) and ethidium homodimer-1 (0.2 M) in PBS with 1% BSA to discriminate live from dead cells where viability ranged from 5075%. In live cells the nonfluorescent calcein AM is converted to a green-fluorescent calcein after acetoxymethyl ester hydrolysis by intracellular esterases. This dye is also available as 1 mg of the solid (C-1430) and resuspended in DMSO (C-3099). The Live-Dead Cell Viability Assay Kit is a quick and simple three-color assay to measure cell viability. The kit consists of Calcein-AM (stains live cells), Propidium Iodide (stains dead cells) and Hoechst 33342 (stains all cells). The Live Dead assay staining solution is a mixture of two fluorescent A confocal microscope (SP 8, Leica) was used to image live cells with excitation/emission at 495 nm/515 nm and dead cells at 495 nm/635 nm, respectively. [email protected]@[email protected] NPs to kill HFLS-RA in vitro was determined using the calcein-AM//PI assay. Surface staining of mouse splenocytes combined with LIVE/DEAD Fixable Aqua Dead Cell Stain (ThermoFisher Scientific). The plate was incubated at 37C and 5% CO 2 for three time points: 1, 3 and 7 days. Five different live/dead assays were investigated (fluorescein diacetate (FDA)/propidium iodide (PI), Syto 9/PI (BacLight), FDA/Sytox red, Calcein acetoxymethyl (AM)/Sytox red, and carboxyfluorescein diacetate (CFDA)/Sytox red). In live cells the nonfluorescent calcein AM is converted to a green-fluorescent calcein after acetoxymethyl ester hydrolysis by intracellular esterases. Most chondrocytes were alive at different time points, except for a very small number dying on day 5, as shown by the live/dead staining ( Fig. Sensitive detection of fluorescent probes aid in evaluating cell signaling and death Add Calcein AM to the buffer in a final concentration of 15M for fluorescent labeling, mix well 3. Incubate 37oC for 30min with occasional shaking 4. To further evaluate cell viabilities of 4T1 after different treatments, calcein acetoxymethyl ester (calcein-AM) and propidium iodide (PI) was utilized to stain the live and dead cells. Calcein AM-based assays can be used in adherent or suspension cultures of eukaryotic cells, spheroid or 3D-cell culture models, and certain live tissue preparations; download the Reference List for examples. The two probes in the kit eflect cell viability by measuring intracellular esterase activity and plasma membrane integrity. Live/dead assay . (D) After pretreatment (as described in Fig. (green: living cells; red: dead cells). Calcein and ethidium staining was used to label live and dead CMs immediately after isolation. The non-fluorescent calcein AM permeates the intact cell membrane and is converted into calcein, the fluorescent form, by intracellular esterases. Scale bar, 400 m. f, Monitoring CEx fusion by cobaltcalcein assay; calcein fluorescence ( ex = 480 nm and em = 510 nm) versus time (010 min) with/without ZERO BIAS - scores, For the Live/Dead staining assay, the cells were incubated with calcein-AM/propidium iodide (Beyotime, China) for 30 min and examined using a fluorescence microscope. BMM live/dead assays. [email protected]@[email protected] NPs to kill HFLS-RA in vitro was determined using the calcein-AM//PI assay. Phototheranostics is a potential area for precision medicine, which has received increasing attention for antibacterial applications. The living and dead cells were stained with a live/dead assay (Calcein-AM/PI Double Staining Kit) after 24 h of culture. Live cells and tissues are imaged over long time periods using a fast, non-toxic click chemistry. Scale bar, 100 m. Calcein and ethidium staining was used to label live and dead CMs immediately after isolation. f, Monitoring CEx fusion by cobaltcalcein assay; calcein fluorescence ( ex = 480 nm and em = 510 nm) versus time (010 min) with/without (C) K7M2 cells were pretreated with the autophagy inhibitor 3-MA (5 mM), and then incubated with CZP for 4 d to determine the cell viability. Viability/Cytotoxicity Assay Kit for Animal Live & Dead Cells (Calcein AM, PI method) provides dual fluorescence staining for the detection of living and dead cells. Calcein and Propidium Iodide Assay Protocol: The calcein assay is based on the conversion of the cell permeant non-fluorescnt calcein AM dye to the fluorescent calcein dye by intracellular esterase activity in live cells. The application of chemodynamic therapy (CDT) for cancer is a serious challenge owing to the low efficiency of the Fenton catalyst and insufficient H2O2 expression in cells. The Live and Dead Cell Assay Kit (Calcein AM, 7-AAD) kit combines Calcein AM with 7-aminoactinomycin D (7-AAD) to allow for easy and simultaneous labeling of live, membrane compromised, and dead cells within a single sample. The proteins of both RBCm and Mm were quantified using the BCA Protein Assay Kits (Solarbio Life Sciences). In brief, the lives cells were stained by Calcein AM to show green fluorescence while dead cells were dyed by PI to emit bright red fluorescence. AAT Bioquest Live or Dead Cell Viability Assay Kits are based on the simultaneous determination of live and dead cells using two fluorescent probes. After 24 or 72 h incubation, the treated cells were stained with a LIVE/DEAD viability assay kit (10 g/mL) for 20 min, FeRhoNox-1 (10 g/mL) for 1 h, C11-BODIPY (581/591) (10 mM) reagent for 1 h, or 100 M Pimonidazole Hydrochloride as well as a fluorescent antibody (FITC-MAb1) based on a reported method . The easiest method is to do fluorescence microscopy and then counting the dead cells and live cells in the population by visual observation or by ImageJ. CBA415 Molecular formula :C 46 H 46 N 2 O 23. The Live cell dye labels intact, viable cells green. The LIVE/DEAD Viability/Cytotoxicity Kit is a quick and easy, two-color assay to determine viability of cells in a population. The Cell Health Assay is a fluorescence-based assay used for live/dead cell counting that is amenable to fluorescence microscopy and flow cytometry analyses wherein live cells are detected by the conversion of calcein-AM to calcein and dead cells by uptake of propidium iodide. Epub 2011 Feb 9. Zhou S, Cui Z, Urban J. Biotechnol J. In brief, the lives cells were stained by Calcein AM to show green fluorescence while dead cells were dyed by PI to emit bright red fluorescence. Live/Dead PTX PTX Live and Dead Cell Assay Kit (Calcein AM, 7-AAD) (ab270789) combines Calcein AM with 7-aminoactinomycin D (7-AAD) to allow for easy and simultaneous labeling of live, membrane compromised, and dead P values, two Quick and easy to use, the kit allows discrimination between live and dead cells with two probes that measure recognized parameters of cytotoxicity and cell viabilityintracellular esterase activity and plasma membrane integrity. treatment) to determine the background signal from dead cells for both calcein and PI assays. Calcein AM is a widely used live-cell marker. Therefore, a method was used, first described by Poole et al., who stained porcine corneas using the Live/Dead-Assay (Molecular Probes Inc.). The Viability/Cytotoxicity Assay Kit for Animal Live & Dead Cells contains two dyes (calcein AM and EthD-III) for distinguishing live and dead cells. The assay employs two probes that detect intracellular esterase activity in live cells and compromised plasma membrane integrity in dead cells. Product Information. As shown in Fig. The non-fluorescent calcein AM dye is hydrolyzed by cellular esterases to give calcein which is fluorescent and is retained in the cytoplasm. Calcein-AM, acetoxymethyl ester of calcein, is highly lipophilic and cell membrane permeable. Cell viability was determined with a live/dead viability/cytotoxicity kit for mammalian cells (Thermo Fisher Scientific) by adding 4 M calcein and Additional resources Indicates live, dead and total cell count. H 2 induced apoptosis through mitochondria-dependent pathways. The Live/Dead Assay Kit provides a two-color fluorescence assay that detects both live and dead cells for mammalian cell types. To determine the expression of caspase-3 in cells, specimens were fixed with 4% (v/v) formalin solution Specifically, 4T1 cells were inoculated in a 12-well plate (1 10 5 cells per well). Next, a live/dead staining assay was used to visually evaluate the cell biocompatibility. For a longer wavelength version of this dye, check out our new CellTrace calcein red-orange AM (C-34851). A bacterial viability/cytotoxicity assay is also available at PromoKine (PK-CA707-30027). After being cultured for 24 h, the cell-laden MSNF/Gel scaffolds were washed with DPBS three times and treated with ethidium homodimer-1 (0.5 M) and Calcein AM (0.25 M) for 45 min. Cells were treated with calcein AM dye and ethidium homodimer-1 (Invitrogen) at a final concentration of 1.33 M calcein AM and 2.5 M ethidium homodimer-1. The Live/Dead Cell Double Staining Kit is utilized for simultaneous fluorescence staining of viable and dead cells. The aim of this protocol is to provide instructions for performing live dead staining using Calcein AM and Propidium iodide (PI) for imaging of live respectively dead cells in 3D constructs of CELLINK bioink, GelMA and GelX variations. (E) CLSM images of the K7M2 cells using calcein-AM/propidium iodide (CAM/PI) double staining kit. To investigate the biocompatibility of these MSNF/Gel scaffolds, Live/dead viability (Molecular Probes) assay was performed. A phenanthroline assay was performed to determine the iron concentrations in PI and GPI NPs . Live/dead stain was prepared by adding 2 mol/L acetomethoxy derivate of calcein (calcein-AM) and 2 mol/L ethidium homodimer-1 per Herein, we fabricated a PDGFB targeting, biodegradable FePt alloy assembly for magnetic resonance imaging (MRI)-guided chemotherapy and starving-enhanced chemodynamic therapy for cancer The retention of Calcein within live cells is dependent upon the inherent properties of the cell type and culture conditions. Prepare the calcein acetoxymethyl/ethidium homodimer-1, 2X (2X calcein AM/EthD-1) reagent fresh at the time of the assay. Here, a type of quaternary amine The fluorescence intensity is proportional to esterase activity, thereby this assay is used to evaluate the cell viability. They are incredibly versatile, and can be designed to measure virtually any cellular or biochemical function. Caco-2 cells were seeded in 12-well plates and cultured for 3 days. The dead cell dye is generally membrane impermeant and therefore cannot pass through intact cell membranes. Calcein AM is a widely used live-cell marker. Calcein AM Calcein AMCalcein494nm517nmPI-DNA535nm617nm Calcein AM Dye is more robust than tetrazolium salt or AlamarBlue Dye as the cells can be stained and quantified in less than 2 hrs. Integrating all phototheranostic modalities in a single molecule and achieving precise spatial colocalization is a challenging task because of the complexity of energy dissipation and molecular design. One example for a functional assay is the commonly known and widely used live dead assay. Ethanol is flammable. 5 a). The LIVE/DEAD Viability/Cytotoxicity Kit is a quick and easy two-color assay to determine viability of cells in a population based on plasma membrane integrity and esterase activity. Advantages Over Alternative Methods Include: Faster This dye is also available as 1 mg of the solid (C-1430) and resuspended in DMSO (C-3099). Live/dead staining and CCK8 test of chondrocytes were conducted after incubation in the leaching solution prepared as described above for 1, 3, and 5 days. Live/Dead assay Live/Dead assays often rely upon both the membrane integrity and either esterase or metabolic activity of live populations and involve the use of two dyes, one that stains live populations, and one that stain dead populations. For a longer wavelength version of this dye, check out our new CellTrace calcein red-orange AM (C-34851). Bioz Stars score: 99/100, based on 1 PubMed citations. The images were taken by an inverted fluorescence microscope. A Calcein AM and PI staining showed confocal fluorescence images of MCF-7 cells with or without xenon lamp irradiation for 10 min. For the Live/Dead staining assay, the cells were incubated with calcein-AM/propidium iodide (Beyotime, China) for 30 min and examined using a fluorescence microscope. Thermo Fisher calcein am ethidium homodimer live dead assay kit Calcein Am Ethidium Homodimer Live Dead Assay Kit, supplied by Thermo Fisher, used in various techniques. The LIVE/DEAD Violet Viability/Vitality Kit provides a two-color fluorescence assay based on the measure of two essential cell health parameters: plasma membrane integrity to measure dead cells and intracellular esterase activity to measure live cells (Figure 8). In live cells the nonfluorescent calcein AM is converted to a green-fluorescent calcein after acetoxymethyl ester hydrolysis by intracellular esterases. A confocal microscope (SP 8, Leica) was used to image live cells with excitation/emission at 495 nm/515 nm and dead cells at 495 nm/635 nm, respectively. Calcein AM is a widely used live-cell marker. This kit enables the detection of both the live and dead populations of mammalian cells based on the integrity of the cell membrane using a fluorescence microplate reader. (E) CLSM images of the K7M2 cells using calcein-AM/propidium iodide (CAM/PI) double staining kit. Live Dead Cell Viability Assay Kit for 3D and 2D cell culture CBA415 Sigma-Aldrich Live Dead Cell Viability Assay Kit for 3D and 2D cell culture Download Zoom Live-dead cell viability kit for 3D and 2D cell cultures. Calcein AM Calcein AMCalcein494nm517nmPI-DNA535nm617nm Phototheranostics is a potential area for precision medicine, which has received increasing attention for antibacterial applications. Live/Dead staining can be performed using a microscopy-based assay or a high-throughput, microplate-based assay. In live cells the nonfluorescent calcein AM is converted to a green-fluorescent calcein after acetoxymethyl ester hydrolysis by intracellular esterases. The Live/Dead stainning test was conducted with calcein/propidium iodide (Calcien/PI) (Beyotime Institute of Biotechnology, China). Bioz Stars score: 99/100, based on 1 PubMed citations. 2B), cell death was quantified by trypan blue exclusion assay. For microscopy: (1) Prepare pads for imaging - Dilute 1 mM stock solution of calcein-AM (in DMSO) 1:1000 into molten 1% agar prepared using desired D. discoidium growth medium. (C) K7M2 cells were pretreated with the autophagy inhibitor 3-MA (5 mM), and then incubated with CZP for 4 d to determine the cell viability. Prevent entry into sewers, water courses, basements or confined areas. See our full line of Cell Viability and Apoptosis Assays for fixed cell assays and fixable dead cell stains. Cells were resuspended in 300 l of calcein AM (0.2 M) and ethidium homodimer-1 (0.2 M) in PBS with 1% BSA to discriminate live from dead cells where viability ranged from 5075%. P values, two Here, a type of quaternary amine (D) After pretreatment (as described in Fig. The proteins of both RBCm and Mm were quantified using the BCA Protein Assay Kits (Solarbio Life Sciences). Test Conditions A. In live cells the nonfluorescent calcein AM is converted to green-fluorescent calcein, after acetoxymethyl ester hydrolysis by intracellular esterases. An ideal cell viability assay should be safe, rapid, reliable, efficient, and time- and cost-effective, and should not interfere with the test compound. This dye is also available as 1 mg of the solid (C-1430) and resuspended in DMSO (C-3099). As shown in Fig. A phenanthroline assay was performed to determine the iron concentrations in PI and GPI NPs . These fluorescent-based LIVE/DEAD assays can be used to evaluate viability in cellular populations, bacteria, fungi, and yeast. H 2 induced apoptosis through mitochondria-dependent pathways. Live/dead staining and CCK8 test of chondrocytes were conducted after incubation in the leaching solution prepared as described above for 1, 3, and 5 days. Dead cell counts during serum cultivation are underestimated by the fluorescent live/dead assay. Note that Calcein AM-based assays can be used in adherent or suspension cultures of eukaryotic cells (3) and certain tissues (4), but cannot be used in yeast or bacteria. Move containers from spill area. It is membrane permeant and non-fluorescent until ubiquitous intracellular esterases remove ester groups and render the molecule fluorescent. Material needed - Stock solution of 1 mM Calcein AM* - Stock solution of 2 mg/ml PI** - Scale The LIVE/DEAD Violet Viability/Vitality Kit provides a two-color fluorescence assay based on the measure of two essential cell health parameters: plasma membrane integrity to measure dead cells and intracellular esterase activity to measure live cells (Figure 8). This dye is also available as 1 mg of the solid (C-1430) and resuspended in DMSO (C-3099). Quick and easy to use, the kit allows as determined by the enzymatic conversion of virtually nonfluorescent cell-permeant calcein AM to intensely fluorescent calcein, which is well-retained within live cells; Calcein AM or Calcein acetoxymethyl ester is a hydrophobic compound which passes easily through cell membrane into live cells and is used for cell viability assays. Surface staining of mouse splenocytes combined with LIVE/DEAD Fixable Aqua Dead Cell Stain (ThermoFisher Scientific). Since dead cells lack esterase activity, only live cells are labeled and detected. The cell viability was determined by a LIVE/DEAD viability/cytotoxicity kit for mammalian cells (Thermo Fisher Scientific) by adding 4 M calcein and ethidium homodimer-1 into the culture media. To determine the cell viability, BMMs were incubated with spiky particles or rough particles at a dose of 0.005, 0.01, 0.02, 0.04 or (D) Livedead staining of HeLa cells subjected to the various treatments. The non-fluorescent calcein AM permeates the intact cell membrane and is converted into the fluorescent calcein by intracellular esterases. After 24 or 72 h incubation, the treated cells were stained with a LIVE/DEAD viability assay kit (10 g/mL) for 20 min, FeRhoNox-1 (10 g/mL) for 1 h, C11-BODIPY (581/591) (10 mM) reagent for 1 h, or 100 M Pimonidazole Hydrochloride as well as a fluorescent antibody (FITC-MAb1) based on a reported method . Do not place near open flame or heat. Viability/Cytotoxicity Assay Kit for Animal Live & Dead Cells (Calcein AM, EthD-I method) provides dual fluorescence staining for the detection of living and dead cells.The two probes in the kit eflect cell viability by measuring intracellular esterase activity and plasma membrane integrity. Live/dead cell viability assay. Cell viability was determined with a live/dead viability/cytotoxicity kit for mammalian cells (Thermo Fisher Scientific) by adding 4 M calcein and Most chondrocytes were alive at different time points, except for a very small number dying on day 5, as shown by the live/dead staining ( Fig. The EarlyTox Live/Dead Assay Kit contains two markers for live or dead cells that are suitable for use with mammalian cells. Calcein AM, 4 mM in anhydrous DMSO (component of EarlyTox Live/Dead Assay Kits and EarlyTox Live cell Assay Kits) Section 6. Cas number: 148504-34-1. 3. Two fluorophores are utilized to label live cells and dead cells. Live/Dead reagent stock solutions: Calcein AM, Ethidium Homodimer (EthD-1) Safety 1. Lack of red-colored cells and the presence of cells stained in green indicate the lack of toxicity (D). o Calcein AM stock = 1.005 mM (1 mg/ml; aliquots are stored @ -20C) The linear range of the Live/Dead Assay is approximately 500 to 106 cells. The live cells were stained with 2 M calcein-AM for 20 min, while the dead cells were stained with 4.5 M propidium iodide (PI) for 20 min. This dye is also available as 1 mg of the solid (C-1430) and resuspended in DMSO (C-3099). In live cells the nonfluorescent calcein AM is converted to a green-fluorescent calcein after acetoxymethyl ester hydrolysis by intracellular esterases. Wear disposable latex gloves at all times 2. The images were taken by an inverted fluorescence microscope. The living and dead cells were stained with a live/dead assay (Calcein-AM/PI Double Staining Kit) after 24 h of culture. C Fluorescence quantification of ROS. Integrating all phototheranostic modalities in a single molecule and achieving precise spatial colocalization is a challenging task because of the complexity of energy dissipation and molecular design. Storage: -20C; protect from light. Preparation for the Assay 3. To investigate the biocompatibility of these MSNF/Gel scaffolds, Live/dead viability (Molecular Probes) assay was performed. To determine the expression of caspase-3 in cells, specimens were fixed with 4% (v/v) formalin solution Thermo Fisher Scientific LIVE/DEAD cell viability assays provide for easy and sensitive differentiation of live, dead, and cytotoxic populations using fluorescence microscopy, flow cytometry, or microplate readers . Cell viability was characterized by the LIVE/DEAD Cell Imaging Kit (ThermoFisher Scientific). Caco-2 cells were seeded in 12-well plates and cultured for 3 days. The LIVE/DEAD Cell Imaging Kit is a sensitive two-color fluorescence cell viability assay optimized for FITC and Texas Red filters. The Live/Dead Cell Staining Kit II utilizes two fluorescent dyes, Calcein-AM and Ethidium homodimer III (EthD-III).Cellular assays have always been a powerful tool in the research lab. 2.4 Using samples of dead cells, select a calcein AM concentra-tion that does not give significant fluorescence in the dead cell cytoplasm (try from 0.1 to 10 M calcein AM). After being cultured for 24 h, the cell-laden MSNF/Gel scaffolds were washed with DPBS three times and treated with ethidium homodimer-1 (0.5 M) and Calcein AM (0.25 M) for 45 min. Living/dead staining and apoptosis assay. The cells were treated with DMEM medium containing different samples for 24 hours, and the live/dead cells were stained with calcein AM and propidium iodide. The Cell Health Assay is a fluorescence-based assay used for live/dead cell counting that is amenable to fluorescence microscopy and flow cytometry analyses wherein live cells are detected by the conversion of calcein-AM to calcein and dead cells by uptake of propidium iodide. In live cells the nonfluorescent calcein AM is converted to green-fluorescent calcein, after acetoxymethyl ester hydrolysis by intracellular esterases. Live cells are identified using either a calcein acetoxymethyl (AM) derivative or a mitochondrial membrane potential probe, while dead cells are labeled using a cell-impermeable DNA binding dye. B ROS changed in MCF-7 cells after hydrogen treatment. This dye is also available as 1 mg of the solid (C-1430) and resuspended in DMSO (C-3099). Cell viability was characterized by the LIVE/DEAD Cell Imaging Kit (ThermoFisher Scientific). The cells were treated with DMEM medium containing different samples for 24 hours, and the live/dead cells were stained with calcein AM and propidium iodide. ZERO BIAS - scores, After 120 min of oral exposure, analysis was performed with an epifluorescence microscope. For flow cytometry, cells were washed once in PBS and resuspended in 1mL of calcein (LIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells, Molecular Probes, OR, USA) diluted 1/80 in DMSO and 5L Propidium Iodide (PI) at 0.5mg/mL. The Live/Dead stainning test was conducted with calcein/propidium iodide (Calcien/PI) (Beyotime Institute of Biotechnology, China). Dead cells, 3 wells C. Mix of live and dead cells, 3 wells Seed Cells (Day 1) The kit can be used in flow cytometry, fluorescence microscopy, and with fluorescence microplate readers. Sufficient for 5 x 24-well plates or 12 x 96-well plates. To further evaluate cell viabilities of 4T1 after different treatments, calcein acetoxymethyl ester (calcein-AM) and propidium iodide (PI) was utilized to stain the live and dead cells.