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 cell number. 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.

Absorbance Assays

• Quantitate cells based on their metabolic activity
• Simple, homogeneous assays
• Usually require several hours development time
• Compounds like antioxidants can interfere with assays

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. Available with blue, green, and red fluorescence.

MitoView™ Dyes

Loss of mitochondrial membrane potential is a hallmark for apoptosis. Biotium offers MitoView™ 633 dye for membrane potential-sensitive staining of mitochondria by microscopy or flow cytometry. For simultaneous detection of mitochondrial membrane potential and caspase-3 activity, see the NucView®488 and MitoView™633 Apoptosis Assay.

We also offer MitoView™ 405, which changes localization upon mitochondrial depolarization, and MitoView™ Green, a membrane-potential independent mitochondrial dye that can be used to image mitochondria following mitochondrial depolarization, or after fixation.

JC-1 and Other 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.

Viability/Cytotoxicity Assay Kit

The Viability/Cytotoxicity Assay Kit for Animal Live & Dead Cells stains live cells with green fluorescence and dead cells with red fluorescence for detection by fluorescence microscopy or flow cytometry. Calcein-AM is a non-fluorescent cell-permeable ester that can passively enter cells. In viable cells, it is hydrolyzed by esterases in the cytoplasm to release the green fluorescent dye calcein. Calcein itself is membrane-impermeant and is only retained in viable cells with intact plasma membranes. Ethidium Homodimer III is a red fluorescent membrane-impermeant nucleic acid dye. It selectively stains dead cells with damaged plasma membranes (necrotic or late apoptotic cells).

Dead Cell Nucleic Acid Stains

Membrane-impermeant nucleic acid dyes can be used to selectively stain necrotic and late apoptotic cells that have leaky plasma membranes. These dyes have very low fluorescence until they bind DNA or RNA, so they can be used to stain cells without a wash step for homogenous and real-time imaging in cell culture medium. Biotium offers dead cell stains in a variety of colors, as well as Apoptosis & Necrosis Staining Kits with dead cell stains and apoptosis markers.

Live-or-Dye™ Fixable Dead Cell Stains

Live-or-Dye™ Fixable Viability Stains are designed for discrimination between live and dead cells. The dyes are cell membrane impermeable and amine-reactive. They enter dead cells with damaged plasma membranes and covalently label intracellular proteins. The dyes react with surface proteins on live cells, but because these are much less abundant than intracellular proteins the staining is very low. Live-or-Dye™ labeling is extremely stable, so cells can be fixed and permeabilized without loss of fluorescence or dye transfer between cells. Biotium offers a selection of 14 bright and photostable Live-or-Dye™ stains for maximal flexibility in multi-color analysis.

Flow Cytometry

Live/dead stains are useful probes to include when analyzing cell surface protein expression by flow cytometry, because they allow intracellular fluorescence signal from dead cells with permeable plasma membranes to be excluded from analysis. In addition, several Live-or-Dye™ stains were designed specifically for spectral flow to fill in gaps between common fluorophores: Live-or-Dye™ 510/550, 665/685, 375/600, and 615/740.

Fluorescence Microscopy

The staining protocol has been optimized to maximize live/dead discrimination with minimal live cell staining, in order to prevent interference with immunostaining. In microscopy, live/dead stains allow unambiguous visual discrimination of dead cells.

NucView® Caspase Substrates for Live Cells

NucView® Caspase-3 Substrates are novel fluorogenic substrates developed by Biotium to detect apoptosis in intact cells in real-time. Biotium has invented a new concept in the design of fluorogenic enzyme substrates. The enzyme substrate is attached to a fluorogenic DNA dye. 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 to emit fluorescence. In the case of NucView® Caspase-3 Substrates, the caspase-3/7 substrate peptide DEVD is attached to a DNA-binding dye. The substrate enters the cell cytoplasm where it is cleaved by caspase-3 in apoptotic cells to release the fluorogenic DNA dye, which stains the nucleus. Unlike FLICA assays that use irreversible inhibitors to label active caspases, NucView® caspase-3 substrates do not interfere with caspase activity, allowing monitoring of caspase activity in real time. 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.

Other Caspase Substrates and Assays

In addition to our patented NucView® technology for detecting caspase-3 activity in live cells, Biotium also offers rhodamine 110 (R110)-based assay kits for fluorescence- or absorbance-based detection of caspase-3 or caspase-8 activity in cell lysates. The HTS versions of the R110-based homogenous caspase-3 and caspase-8 assay kits are optimized for high throughput screening by fluorescence microplate reader. We also offers a coumarin (AMC)-based blue fluorogenic substrate (Ac-DEVD-AMC) for measuring caspase-3 activity in cell lysates by fluorescence microplate reader.

Apoptosis Inducers/Inhibitors

Staurosporine is a broad range protein kinase inhibitor that induces apoptosis in cultured cells. It is useful as a positive control for many apoptosis assays. We also offer ionomycin, a calcium ionophore that has been shown to induce apoptosis through calpain activation. Caspase inhibitor Ac-DEVD-CHO is a competitive inhibitor of caspase-3 for use in cell lysates.

Dual Apoptosis & Necrosis Assay Kits

Annexin V is a 36 kDa 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 our bright and photostable 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 fluorogenic nucleic acid dyes that stain necrotic and late apoptotic cells with damaged plasma membranes. EthDIII is a novel red fluorescent dead cell stain with higher affinity and quantum yield compared to PI, while RedDot™2 is a far-red dead cell stain for the Cy®5 channel. 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 .

Annexin V conjugates are available with a wide selection of CF® dyes and other labels, including Near-IR CF® Dye Annexin V conjugates. We offer dead cell nucleic acid stains separately as well, including far-red fluorescent RedDot™2 far-red dye and green fluorescent NucSpot® 470 stain. Note: Apoptosis & Necrosis Staining kits and Annexin conjugates cannot be used with fixed cells or tissues. For fixed cell stains, see our TUNEL Assays.

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.