Cell Viability and Apoptosis

Absorbance-Based Cell Quantitation Assays

MTT, XTT, and resazurin are reduced by active mitochondria to yield colored products, and thus are useful for assaying cell viability and quantitating cells. MTT and XTT are reduced to colored formazin salts that can be measured by absorbance. Resazurin is reduced to form resorufin, which can be measured by either absorbance or fluorescence. These are easy-to-use homogeneous assays, but they have the disadvantage of requiring several hours for development. In addition, drugs or compounds with redox activity, like antioxidants, can interfere with the assays by directly affecting substrate reduction. MTT assay requires cell lysis before the absorbance can be measured. XTT and resazurin do not require cell lysis, allowing measurements to be made at multiple timepoints.

alamarBlue is a registered trademark of Trek Biosystems. CellTiter-Blue is a registered trademark of Promega Corporation.

Steady-ATP™ HTS Cell Viability Assay

Steady-ATP™ HTS Viability Assay Kit allows highly sensitive quantitation of cellular ATP levels for viability assessment, with the ability to detect as low as 16 cells per well in a 384-well plate. The assay offers a robust signal half-life of over 5 hours, and a linear signal of 3-4 orders of magnitude. The procedure is designed with convenience in mind, requiring only a single addition of the homogeneous Steady-ATP™ Assay Mix directly to cells in cultured medium, with no washing or medium exchange required.

ViaFluor® SE Cell Proliferation Kits

ViaFluor® SE Cell Proliferation Kits can be used to monitor cell division by flow cytometry. ViaFluor® SE dyes are membrane-permeant compounds that are initially non-fluorescent esters, but are converted to fluorescent dyes by intracellular esterases and will covalently react with amine groups on intracellular proteins at the same time, forming fluorescent conjugates that are retained in the cell. Immediately after staining, a single bright fluorescent population will be detected by flow cytometry.

With each cell division, daughter cells inherit roughly half of the fluorescent label, allowing the number of cell divisions that occur after labeling to be detected by the appearance of successively dimmer fluorescent peaks on a flow cytometry histogram compared to cells analyzed immediately after staining. Cell proliferation dyes can be used to track cell divisions in vivo or in vitro. The staining also can withstand fixation and permeabilization for subsequent immunostaining.

Pacific Blue and Texas Red are registered trademarks of Thermo Fisher Scientific.

Classic Mitochondrial Membrane Potential Dyes

In healthy cells, JC-1 dye aggregates in mitochondria as a function of membrane potential, resulting in red fluorescence with brightness proportional to the membrane potential. Conversely, in apoptotic and necrotic cells with diminished mitochondrial membrane potential, JC-1 exists in a green fluorescent monomeric form in the cytosol, allowing of cell viability to be assessed by measuring the ratio of red to green fluorescence by flow cytometry or fluorescence microplate reader. Rhodamine 123 is a green fluorescent mitochondrial membrane potential dye. Red fluorescent TMRM and TMRE are the preferred dyes for quantitative membrane potential measurements.

Draq7 is a trademark of Biostatus, Ltd. Texas Red and Pacific Blue are registered trademarks of Thermo Fisher Scientific. Cy Dye is a registered trademark of Cytiva.

NucView® Caspase Substrates for Live Cells

NucView® Caspase-3 Substrates are enzyme substrates attached to a fluorogenic DNA dye to detect apoptosis in intact cells in real-time. Before cleavage, the dye is non-fluorescent and unable to bind to DNA. Upon enzymatic cleavage of the substrate, the dye is released and becomes capable of binding to DNA. The caspase-3/7 substrate peptide DEVD is attached to a DNA-binding dye and when it enters the cell cytoplasm where it is cleaved by caspase-3 in apoptotic cells, the fluorogenic DNA dye is released and stains the nucleus. Unlike FLICA assays, NucView® caspase-3 substrates do not interfere with caspase activity. NucView® can be fixed with formaldehyde after staining, but it cannot be used to stain fixed cells or tissues. For staining of fixed cells or tissues, see our TUNEL Assays.

Pacific Blue is a registered trademark of Thermo Fisher Scientific. Cy Dye is a registered trademark of Cytiva.

Dual Apoptosis & Necrosis Assay Kits

Annexin V is a protein that has high affinity for phosphatidylserine (PS). During apoptosis, PS is translocated from the inner to the outer leaflet of the plasma membrane, where it can be stained by fluorescent conjugates of Annexin V. We offer kits with CF®488A Annexin V and a variety of dead cell nucleic acid stains, including propidium iodide (PI), 7-AAD, Ethidium Homodimer III (EthDIII), and RedDot™2. These are membrane-impermeant nucleic acid dyes that stain necrotic and late apoptotic cells. The Apoptotic, Necrotic, and Healthy Cells Quantitation Kit Plus also includes blue fluorescent Hoechst DNA dye for visualizing all cells by fluorescence microscopy. Also see our apoptosis and necrosis staining kit with NucView®488 Caspase-3 Substrate and the far-red dead cell stain RedDot™2 .

Cy Dye is a registered trademark of Cytiva.

CF® Dye Fluorescent TUNEL Staining Kits

TUNEL (terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling) is highly selective for the detection of apoptotic cells, but not necrotic cells or cells with DNA strand breaks resulting from irradiation or drug treatment. In this assay, TdT enzyme catalyzes the addition of labeled dUTP to the 3’ ends of cleaved DNA fragments. Fluorescent dye-conjugated dUTP can be used for direct detection of fragmented DNA in fixed cells or tissue sections. We offer TUNEL kits with dUTP conjugates of our bright and photostable CF® Dyes with green, red, or far-red fluorescence. We also offer CF® Dye dUTP Conjugates in a variety of colors.

PathoGreen™ and Other Neuronal Histofluorescent Stains

PathoGreen™ Histofluorescent Stain is an anionic green fluorescent dye functionally similar to Fluoro-Jade® dyes. These dyes stain degenerating neurons and their processes in tissue sections and cultured cells. The mechanism of staining has not been determined, but it is proposed that the negatively charged dyes bind to positively charged polyamines or other molecules generated in dying neurons in response to a variety of different neurotoxic insults. We also offer stains for the detection of amyloid in live or fixed neuronal tissues, including Congo Red, DCDAPH, and Thioflavin T.

Texas Red is a registered trademark of Thermo Fisher Scientific. Cy Dye is a registered trademark of Cytiva.