Custom Premix Selecting "Custom Premix" option means that all of the beads you have chosen will be premixed in manufacturing before the kit is sent to you.
The powerful combination of quantitative image analysis and flow cytometry in a single platform creates exceptional new experimental capabilities. The following are just a few and many ways in which the Amnis® ImageStreamX and FlowSight imaging flow cytometers can bring more power and insight into your research.
Measurement of NFkB translocation in transgenic T cells which are in contact with antigen presenting cells (APC) and specific peptide. Specific T cell:APC conjugates are identified and translocation of NFkB from the cytoplasm to the nucleus specifically within the T cells is measured in the presence (shown) or absence of peptide.
Co-localization of an antibody-drug conjugate to endosomes or lysosomes
In this experiment we use the capabilities of the ImageStream system to measure the transit of a fluorescently labeled antibody-drug conjugate (ADC) through the cellular endocytic pathway. The high spatial resolution afforded by the ImageStream system allowed accurate measurement of ADC co-localization to endosomes and lysosomes.
DNA condensation and fragmentation are hallmarks of apoptosis. By measuring the area and the intensities of the brightest portions of the nuclear image, the bright, punctate nuclear imagery of apoptotic cells can be distinguished from the evenly stained nuclear imagery of a normal, healthy nucleus. This makes possible the automated identification of apoptoticWhen cells begin to dye by apoptosis there is fragmentation and condensation of the DNA. By measuring the area and the intensities of the brightest portions of the nuclear image, the bright, punctate nuclear imagery of apoptotic cells can be distinguished from the evenly stained nuclear imagery of a normal, healthy nucleus. This makes possible the automated identification of apoptotic nuclear morphology.
Trypanosoma brucei is a parasite which causes African sleeping sickness. Shown in the figure is a study in which nuclear localization of a GFP tagged molecule was quantitated in two samples. The histogram shows the Similarity score for a cross correlation between the GFP image and the DRAQ5 nuclear image for each cell. A high Similarity score indicates that the GFP image and the DRAQ5 image are highly similar (GFP has translocated to the nucleus). A low Similarity score is obtained for untranslocated events.
FRET using the ImageStream for detection of protein-protein interactions
In fluorescence microscopy, light at one wavelength is absorbed by a fluorophore and emitted at a longer wavelength. FRET (Fluorescence or Forster Resonance Energy Transfer) can occur when a second fluorophore is in very close contact such that it can accept the energy from the first fluorophore and emit the light at an even longer wavelength. The efficiency of the transfer is extremely sensitive to the separation distance between the fluorophores and therefore when the emission is detected at the longer wavelength the conclusion is that the fluorophores were within approximately 10 nanometers of each other. When two fluorophores (a donor and acceptor pair) are used to label two different proteins, the close proximity of the two proteins are inferred by the measurement of the FRET from the donor to the acceptor fluorophore. The combination of high speed image acquisition and automated quantitative image analysis with FRET on the ImageStream allows measurement of the spatial location of the protein interaction within the cell or between cell conjugates even for rare subpopulations. Distinguishing intracelluar location of FRET vs. location of the proteins when not exhibiting FRET behavior offers a major advancement in understanding signaling pathways.
As hematopoeitc stem cells differentiate through the erythroid lineage, they shrink and eventually enucleate as they progress towards becoming RBC. They also progressively gain expression of glycophorinA and lose CD71 expression. The extruded nuclei express low levels of glycophorinA on their membranes. This experiment demonstrates the unique ability of the ImageStream to classify cells using a combination of intensity, morphology, and location based parameters.
Identification of Harmful Bacteria and Analysis for Filamentation
Species of bacteria from culture or from contaminated food supplies can be identified and quantitated by analyzing for fluorescence intensity of species specific FISH probes, size, and shape. Filamentation, either due to defective replication or as a response to evade host innate immunity can be measured.
The micronucleus assay is the preferred method for evaluating the genotoxicity of drugs and other chemical agents. This experiment demonstrates the ability of the ImageStream system to rapidly classify apoptotic, bi-nucleated, and micronucleated events within cell populations simply using brightfield and nuclear imagery.Traditionally, cell biologists performing micronucleus assays have had to choose between manual microscopy, which is time-consuming and tedious, and flow cytometry, which can rapidly process large cell populations but lacks the ability to image the nucleus in intact cells. The Amnis® ImageStream system solves both problems by combining flow cytometry with sophisticated digital imaging and analysis capabilities.
HIV-specific nuclear localization of NFAT was measured in HIV-experienced T cells from peripheral blood of HIV+ patients. HIVgag peptide specifically stimulates NFAT (green) to move to the nucleus (red) in HIV-tetramer positive cells (orange). The Similarity score correlates DRAQ5 nuclear stain with the NFAT signal. The higher the Similarity score, the more translocation is visualized in the example images from each quadrant.
Analysis of organisms in wharf tows by shape and size
Unique species of diatoms and other plankton species from culture or from seawater can be identified and quantitated by analyzing for size, shape, fluorescence intensity, texture, and location/co-location of fluorescent labels and/or chloroplasts.
