Flow cytometry has been utilized for decades and continues to be a very useful tool in scientific research and diagnosis. Flow cytometry is a laser-based technology used in cell sorting and analysis in order to measure a particle’s size, granularity, and fluorescence intensity. Using the principles of light scattering, light excitation, and the emission of fluorochrome molecules, specific multi-parameter data points are generated from particles and cells. Flow cytometry allows researchers to measure several physical characteristics of cells including their shape, size, and internal complexity. Previously used for cell counting and cell sorting, researchers are now using flow cytometry to examine a broad range of cell characteristics including surface markers, protein expression, intracellular ions and other biochemical species, DNA content, redox state, and cell functions such as apoptosis, cell cycle, and phosphorylation status.
How does flow cytometry work?
Flow cytometry uses the principle of hydrodynamic focusing to present cells to the lasers. The sample containing various cells is injected into the center of a sheath flow. The flow chamber is conical in shape, as the reduced diameter forces the cells into the center of the stream. As the sample of cells intercepts the light source, light is scattered across the cells and fluorochromes move into a higher energy state as they get excited. These fluorochromes are then artificially conjugated. When analysis is done by flow cytometry, the cells expressing the antibody-specific marker will manifest fluorescence while cells that lack the marker will not. Researchers can use this technique to determine patterns and understand cell behavior in their samples.
Our Flow Cytometry Assays
Enzo Life Sciences offers a variety of flow cytometry-based assays for cell viability, proliferation and death analysis, available in a wide range of formats to meet individual research needs. These flexible assays are compatible with all flow cytometry platforms, regardless of size or functionality. The assays are easy to perform and can be run quickly using small sample input, without sacrificing sensitivity. This portfolio has been designed to include multiple application-driven configurations providing information on different parameters of interest. Below, we highlight three kits featured in our cell viability, proliferation and death, oxidative stress, and drug disposition platforms, respectively.
Our
CYTO-ID® Autophagy Detection Kit measures autophagic vacuoles and monitors autophagic flux in lysosome-inhibited live cells using a novel dye that selectively labels accumulated autophagic vacuoles. The dye has been optimized through the identification of titratable functional moieties that allow for minimal staining of lysosomes while exhibiting bright fluorescence upon incorporation into pre-autophagosomes, autophagosomes, and autolysosomes (autophagolysosomes). The assay offers a rapid and quantitative approach to monitoring autophagy in live cells without the need for cell transfection.
The probe is a cationic amphiphilic tracer (CAT) dye that rapidly partitions into cells in a similar manner as drugs that induce phospholipidosis. Careful selection of titratable functional moieties on the dye prevents its accumulation within lysosomes, but enables labeling of vacuoles associated with the autophagy pathway.
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Figure 1. HeLa cells were subjected to starvation and recovery and then labeled with CYTO-ID®Autophagy dye. The dye enables clear detection and quantification of autophagic and pre-autophagic vacuoles.
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The
ROS®-ID Hypoxia/Oxidative stress detection kit is designed for functional detection of hypoxia and oxidative stress levels in live cells (both suspension and adherent) using fluorescent microscopy or flow cytometry. This kit includes fluorogenic probes for hypoxia (red) and for oxidative stress levels (green) as two major components. The Hypoxia (Red) dye takes advantage of the nitroreductase activity present in hypoxic cells by converting the Nitro group to hydroxylamine (NHOH) and amino (NH2) and releasing the fluorescent probe. The Oxidative Stress Detection Reagent is a non-fluorescent, cell-permeable total ROS detection dye which reacts directly with a wide range of reactive species. The generated fluorescent products can be visualized using a wide-field fluorescence microscope equipped with standard fluorescein (490/525 nm) and Texas Red (596/670 nm) filters, confocal microscopy, or cytometrically using any flow cytometer equipped with a blue (488 nm) laser.
Figure 2. Detection of hypoxia and oxidative stress levels in cultured human HeLa and HL-60 cells. Cells were treated with hypoxia inducer (DFO) and ROS inducer (pyocyanin). Numbers in each quadrant reflects the percentage of cells (population). Results indicate that hypoxia and oxidative stress dye are specific.
The
EFLUXX-ID® Gold Multidrug resistance assay allows for functional detection of all three clinically relevant ABC transporter proteins: MDR1 (p-glycoprotein), MRP1/2, and BCRP. The assay uses a hydrophobic, non-fluorescent compound that readily penetrates the cell membrane, where it is hydrolyzed to a hydrophilic fluorescent dye by intracellular esterases. Unless the EFLUXX-ID® dye is pumped out of the cell, the esterase cleaved dye is trapped inside the cell. Thus, cells exhibiting drug resistance will have diminished fluorescence. EFLUXX-ID® assay is the only available kit for the simultaneous monitoring of all three major ABC reporter proteins with the ability to profile individual pump activity. The proprietary AM-ester form of the EFLUXX-ID® dye is a hydrophobic non-fluorescent compound that readily penetrates the cell membrane and is subsequently hydrolyzed inside of the cells by intracellular esterases. Unless the EFLUXX-ID® dye is pumped out of the cell, the esterase cleaved dye is trapped inside the cell. The fluorescence signal of the dye generated within the cells thus depends upon the activity of the ABC transporters. The cells with highly active transporters will demonstrate lower fluorescence because of the active efflux of the probe from the cell. Application of specific inhibitors of the various ABC transporter proteins allows differentiation between the three common types of pumps.
Figure 3. Detect Activity of all three major ABC transporter proteins. ABC transporter protein activity was evaluated in CHO K1 cells by flow cytometry using eFluxx-ID Green (top), Gold (middle), or Calcein AM (bottom) dyes. Treatment with specific inhibitors of ABC Transporter proteins (shaded histograms) induces retention of dye within cells relative to untreated cells (lined histograms). The difference in mean fluorescence intensity (MFI) is an indication of the corresponding protein activity, as shown by MAF scores [multidrug resistance activity factors], a quantitative measurement of multidrug resistance. Higher MAF scores are a result of superior specificity of eFluxx-ID dyes to specific inhibitors. Calcein AM (a common probe for MDR assays), is unable to detect BCRP activity.
Flow cytometry provides a unique set of capabilities for the analysis of cells and particles. It allows a wide range of application that include molecular assembly, high content analysis of signaling pathways, and multiplexing of biological targets. Enzo Life Science is dedicated to providing new analytical tools for all your Flow Cytometry needs. We offer over 630 antibodies validated for use in flow cytometry. These include isotype controls as well as biotin and fluorescent conjugates for use in evaluating intracellular and extracellular protein. Please contact our
Technical Support Team for further assistance and check out our
10 tips full successful flow cytometry!
