BioActs offers the full spectrum of fluorescent dyes such as FSD FluorTM, Flamma® Fluors, ICG, and Other Dyes,
and also offers Fluorescent Quenchers, Crosslinkers, Fluorescent Antibodies, Bioprobes,
and Microspheres, Magnetic Beads, Dye Labeling Kit, etc.
Apoptosis plays an important role in the pathogenesis of a variety of diseases, such as cancer, myocardial and cerebral ischemia, and neurodegenerative diseases. Since the induction of apoptosis is an essential event in cancer treatments such as chemotherapy and irradiation, in vivo imaging of apoptosis would provide valuable information in monitoring tumor response to different therapies. As an alternative to fluorescent-conjugated proteins or antibodies, small peptide probes may serve as ideal apoptosis imaging probes. They display rapid clearance from blood circulation, efficient tissue penetration, low immunogenicity and cost-effective production. BioActs developed two series of apoptosis specific small peptide based fluorescent probes, ApoFlamma® H and ApoFlamma® PS, for effective monitoring of apoptosis. Both probes might be used alone or in combination with other dyes such as propidium iodide (PI) in order to accurately analyze mixed populations of apoptotic and other cells.
ApoFlamma® H series made up of a covalent-connection of CQRPPR hexapeptide (ApoPep) with corresponding fluorophore. ApoPep strongly binds to apoptotic and necrotic cells but weakly binds to live cells. The receptor for ApoPep was histone H1 that exposed on the surface of apoptotic cells. In necrotic cells, ApoPep entered the cells and bound to histone H1. The monitoring of tumor apoptosis in response to chemotherapy can be easily realized with optical imaging technique utilizing ApoFlamma® H series. We recommend ApoFlamma® H series as versatile fluorescent probes for in vitro and in vivo imaging of apoptosis with targeting for histone H1.
ApoFlamma® PS series enables to selectively detect phosphatidylserine (PS) on the outer leaflet of the plasma membrane. The translocation of PS from the inner to the outer leaflet of plasma membrane occurs in apoptotic cells, and PS serves as most common molecular marker for the detection and measurement of apoptotic cells under both in vitro and in vivo conditions. The PS-recognition moiety in ApoFlamma® PS series is a nonapeptide, with sequence of CLSYYPSYC, and the peptide is labeled with a fluorophore. In animal model study, ApoFlamma® PS series effectively detected tumors yet their binding to other organs such as liver and lung was negligible. BioActs recommends ApoFlamma® PS series as ideal apoptosis detecting probes for both in vitro and in vivo imaging of apoptosis with targeting of PS.
Figure 1. Microscope observation of ApoFlamma® PS series for dead cell
Fluorescence imagings of cells (B16F10, H460, MDA-MB-231): apoptosis was induced by etoposide and then cells were stained with ApoFlamma® PS 675.
Figure 2. Apoptotic cell imaging with ApoFlamma® H FAM
Image was obtained by treating ApoFlamma® H FAM to apoptosis-induced H 460 cell.
Annexin V Flamma® series
In normal live cells, phosphatidyl serine (PS) is located on the cytoplasmic surface of the cell membrane, however PS is translocated to the outer leaflet of the plasma membrane exposing to the external cellular environment when apoptosis is in progress. Annexin V is a Ca2+ dependent PS-binding protein that has been used as a non-quantitative probe to detect cells that have expressed PS on the cell surface. Fluorescence conjugated annexin V enables to detect the externalized PS by binding to PS that exposed on the outer leaflet. BioActs developed a series of Flamma® dyes and other dyes such as FITC, TAMRA and ICG conjugated annexin V, Annexin V-Flamma® series, as PS selective fluorescent probes.
Annexin V Flamma® series is a rapid and selective probe for labeling of externalized PS, an indicator of intermediate stages of apoptosis. Flamma® dyes display strong absorption, high fluorescence quantum yield and high photostability, and they maintain good fluorescence activity and stability after conjugation to biomolecules, allowing the detection of low-abundance biological structures with great sensitivity. Annexin V Flamma® conjugates can be utilized in fluorescent imaging as well as for flow cytometry of apoptotic cells, and they might be used in combination with other dyes such as propidium iodide (PI) in order to accurately analyze mixed populations of apoptotic and other cells. Annexin V Flamma® probes available as stand-alone reagent or easy-to-us kits.
Annexin V Flamma® apoptosis detection kit includes a PI nucleic acid binding dye and annexin V binding buffer. PI is impermeant to live cells and apoptotic cells, but stains dead cells with red fluorescence by binding to nucleic acids. Normal live cell is not labeled by ether Annexin V or PI, early stage apoptotic cell is stained with Annexin V and natural death or cell subject to necrosis is stained with PI. The late stage apoptotic cells are stained by both reagents. After staining a cell population with Annexin V Flamma® and PI in annexin binding buffer, apoptotic cells and dead cells can be distinguished and analyzed using a flow cytometer.
Figure 1. Annexin V Flamma® 488 cell staining
Apoptosis was induced by treating actinomycin D on HL 60 cell, and cell image was obtained after staining with Annexin V Flamma® 488 and PI in 1× Annexin V binding buffer for 15 min. Upper and lower image are same cell. Right image is fluorescence merging image, and the left figures include DIC picture. Cells labeled with green color are stained with Annexin V Flamma® 488, and red stained cells stained by PI.
Flamma® Fluors TUNEL Assay Kit
Apoptosis is a highly regulated and controlled process that confers advantages during an organism's lifecycle, and defective apoptotic processes have been implicated in a variety of diseases such as atrophy and cancers. In situ labeling of apoptotic cells allows highly sensitive and quantitative analysis of involved cells. Characteristics of the late stages of apoptosis are changes in nuclear morphology, including chromatin condensation, degradation of nuclear envelope, and DNA strand breaks. TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling) assays are the most widely used methods for rapid identification and quantification of the apoptotic cell fraction in cultured cell preparations. TUNEL reaction preferentially labels DNA strands that generated during apoptosis using the ability of TdT to label blunt ends of double-stranded DNA independent of a template. This selective labeling allows discrimination of apoptosis from necrosis and from primary DNA strand breaks induced by cytostatic drugs or irradiation.
BioActs developed Flamma® Fluors TUNEL assay kit for quantitative analysis of apoptotic cells in the mixed cell populations. The kit employs dUTP conjugated Flamma® Fluors and FITC dyes for labeling of fixed cells or tissues. Flamma® Fluor dyes display excellent fluorescence activity and photostability after conjugated to biomolecules, allowing detection of low-abundance biological structures with great sensitivity. Flamma® TUNEL assay kit is reliable and effective method to detect and quantify a wide levels of apoptotic cells by fluorescence microscopy and flow cytometry. In addition, the kit can be used in the analysis of apoptotic cell of paraffin-embedded tissue sections along with cultured cells. BioActs offers Flamma® TUNEL assay kit for the detection apoptotic cells in frozen and formalin-fixed tissue sections, determination of the sensitivity of malignant cells to drug-induced apoptosis, discrimination of apoptotic and necrotic cells in the cell death environment, etc.
Components of Flamma® Fluors TUNEL assay kit
|Annexin V Series||Availability :||Reacting Functionality :|
|TUNEL Assay Kit||Availability :||Reacting Functionality :|