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The newer MitoTracker dyes “for flow cytometry” and the original MitoTracker dyes differ only in the packaging. The original MitoTracker dye products are shipped as 20 vials with 50 µg per vial. The newer MitoTracker dyes “for flow cytometry” products are shipped as 5 vials with a proprietary amount of dye per vial. The amount of dye per vial is optimized for flow cytometry experiments. Each vial provides sufficient reagent for 20 flow cytometry experiments.

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This could be due to storage of the product under anaerobic or low oxygen conditions. The Hypoxia Green Reagent for Flow Cytometry increases in fluorescence upon exposure to low oxygen environments, and this change is not reversible. For some ROS indicators, we recommend storing the reagent under dry nitrogen or argon to prevent oxidation during storage. However, this is not appropriate for the Hypoxia Green Reagent for Flow Cytometry.

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When cell purity is the most important criteria, target cell activation is not a concern, or when the downstream applications are isolation of RNA or gDNA, then positive isolation is recommended. When the most important criteria is to isolate cells with minimum disturbance, and where the downstream applications include cell culture, study cell function, morphology, and flow cytometry, then negative isolation is recommended (note that positive isolation of cells can also be used when the downstream applications include cell culture, flow cytometry, or studying cell function, but then a positive isolation with bead release from the cells are required, by using e.g., FlowComp products or the Positive Isolation Kits containing DETACHaBEAD products).

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It is recommended that you first check the product insert for any recommended usage guidelines.

For general purposes, starting concentrations are as follows:
ELISA--0.5-1.0 µg/mL
WB--1-5 µg/mL
IP--2-5 µg per reaction (1 X 10e6 cells)
IHC/IF--1-10 µg/mL
FC--10-20 µg/mL

Please note that these are general guidelines only.

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The following cell health and viability assays can be performed by flow cytometry :

-Apoptosis Assays:
Membrane Asymmetry: Annexin V is a member of a family of structurally related proteins that bind phospholipids in the presence of Ca2+. Annexin V binds several phospholipids, but shows highest affinity for phosphatidylserine.
Phosphatidylserine is normally found in the inner leaflet of the cell membrane; however, in the early stages of apoptosis, phosphatidylserine is observed to translocate to the outer leaflet. This translocation makes phosphatidylserine available for annexin V binding in the presence of Ca2+ containing incubation buffer. Cells undergoing apoptosis will stain with annexin V, while normal cells will not. annexin V is available conjugated with a wide range of fluorophores.

Mitochondrial Health: A distinctive feature of the early stages of apoptosis is the disruption of the mitochondria, including changes in membrane and redox potential. We exclusively offer a number of fluorescent probes for analyzing mitochondrial activity in live cells by flow cytometry, with minimal disruption of cellular function.

The MitoProbe family of mitochondrial stains (MitoProbe DiOC2(3) Assay Kit, Cat. No. M34150, MitoProbe JC-1 Assay Kit, Cat. No. M34152, and MitoProbe DiIC1(5) Assay Kit, Cat. No. M34151) provides quick, easy, and reliable flow cytometric detection of the loss of mitochondrial membrane potential that occurs during apoptosis.

Caspase Activity: The CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit (Cat. No. C10427) enables flow cytometric detection of activated caspase-3 and caspase-7 in apoptotic cells. The kit includes the novel fluorogenic substrate CellEvent Caspase-3/7 Green Detection Reagent which targets the recognition sequence for activated caspase-3 and caspase-7, as well as SYTOX AADvanced Dead Cell Stain.

DNA Fragmentation: The later stages of apoptosis are characterized by changes in nuclear morphology, including DNA fragmentation, chromatin condensation, degradation of nuclear envelope, nuclear blebbing, and DNA strand breaks. DNA fragmentation that occurs during apoptosis produces DNA strand breaks, and can be analyzed using TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays. The APO-BrdU TUNEL assay (Cat. No. A23210) is a two-color assay for labeling DNA breaks and total cellular DNA to detect apoptotic cells by imaging or flow cytometry.

Nuclear Chromatin Condensation: The later stages of apoptosis are characterized by changes in nuclear morphology, including DNA fragmentation, chromatin condensation, degradation of nuclear envelope, nuclear blebbing, and DNA strand breaks. Cells undergoing apoptosis display an increase in nuclear chromatin condensation. As the chromatin condenses, cell-permeable nucleic acid stains becomes hyperfluorescent, thus enabling the identification of apoptotic cells when combined with a traditional dead-cell stain. The Vybrant Apoptosis Assay Kit #5, Hoechst 33342/Propidium Iodide (Cat. No. V13244) provides a rapid and convenient assay for apoptosis based on fluorescence detection of the compacted state of the chromatin in apoptotic cells. The Chromatin Condensation & Membrane Permeability Dead Cell Apoptosis Kit with Hoechst 33342, YO-PRO-1, and PI dyes, for flow cytometry (Cat. No. V23201) detects apoptotic cells with changes in nuclear chromatin condensation and plasma membrane permeability.

-Cell Cycle Analysis:
Live cell assays: The Vybrant DyeCycle family of dyes offers robust fluorescent dyes for live-cell cycle analysis with limited cytotoxicity using 405 nm (Cat. No. V35003), 488 nm (Cat. No. V35004), 532 nm (Cat. No. V35005), or 633 nm (Cat. Nos. V10309 and V10273) excitation. The dyes have low cytotoxicity, allowing stained cells to be sorted and otherwise cultured or assessed with functional assays after staining.

Fixed cell assays: Analyzing cell cycle using FxCycle Violet Stain (Cat. No. F10347), SYTOX AADvanced Dead Cell Stain Kit (Cat. No. S10349) or FxCycle Far Red Stain (Cat. No. F10348) allows for multiple color options for simplified fixed cell cycle analysis.

-Cell Proliferation:
Dye dilution assays for cell proliferation: Dye dilution assays for cell proliferation rely on cell membrane–permeant fluorescent molecules. Upon entry into the cell, the dye will covalently bind to amine groups on proteins, resulting in long-term dye retention within the cell. Through subsequent cell divisions, each daughter cell receives approximately half the fluorescence of the parent. Analysis of the fluorescence intensities of cell populations by flow cytometry enables determination of the number of generations through which a cell or population has progressed since the label was applied. CellTrace fluorescent stains can be used without affecting morphology or physiology to trace generations in vivo or in vitro. There is no known effect on proliferative ability or biology of cells and they are well retained in cells for several days post-stain. Available kits for flow cytometry include CellTrace CFSE Cell Proliferation Kit (Cat. No. C34554), CellTrace Violet Cell Proliferation Kit (Cat. No. C34557), and CellTrace Far Red Cell Proliferation Kit (Cat. No. C34564).

DNA Synthesis Assays: Measuring the synthesis of new DNA is a precise way to assay cell proliferation in individual cells or in cell populations. DNA synthesis–based cell proliferation assays measure the rate of new DNA synthesis based on incorporation of modified nucleosides. The Click-iT Plus EdU cell proliferation assay utilizes the power of click chemistry and the modified nucleoside EdU to provide a superior alternative to BrdU staining for detecting and quantitating newly synthesized DNA. The Click-iT Plus EdU cell proliferation assay is available with Pacific Blue (Cat. No. C10636), Alexa Fluor 488 (Cat. Nos. C10632 and C10633), and Alexa Fluor 647 (Cat. Nos. C10634 and C10635).

-Viability Assays:
Dead cells often give false positive results, as they tend to bind non-specifically to many reagents. Therefore, removing dead cells from your flow cytometry data is a critical step to help ensure accurate results and analysis.

Non-fixable Membrane Permeability Stains: SYTOX Dead Cell Stains (Cat. Nos. S34857, S34860, S34861, S34859, and S34862) do not cross intact cell membranes, and they exhibit increased fluorescence upon dsDNA binding, making them some of our most brilliant dead cell stains. Cell-impermeant classic DNA-binding dyes include propidium iodide (Cat. No. P21493) and 7-AAD (Cat. No. A1310). Both of these dyes have been used extensively for viability assays in flow cytometry. CellTrace Calcein AM dyes can be passively loaded into adherent and nonadherent cells. These cell-permeant esterase substrates serve as viability probes that measure both enzymatic activity, which is required to activate their fluorescence, and cell membrane integrity, which is required for intracellular retention of their fluorescent products. Available with blue (Cat. No. C34853), violet (Cat. No. C34858), and green (Cat. No. C34852) fluorescence, these dyes are ideal for short-term staining of live cells and can be used in multiplexed flow cytometry experiments.

Fixable Viability Stains: The LIVE/DEAD Fixable Dead Cell Stains are fixable viability dyes that help to ensure accurate assessment of cell viability in samples after fixation and/or permeabilization. LIVE/DEAD Fixable Dead Cell Stain Kits are based on the reaction of a fluorescent reactive dye with cellular proteins (amines). These dyes cannot penetrate live-cell membranes, so only cell-surface proteins are available to react with the dye, resulting in dim staining. The reactive dye can permeate the damaged membranes of dead cells and stain both the interior and exterior amines, resulting in more intense staining. LIVE/DEAD Fixable Dead Cell Stain Kits are available in eight single-channel colors available for UV, 405, 488, 532, 561, or 633 nm lasers in three packaging sizes to match your experiment.

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-Calcium flux: Each of the Oregon Green calcium indicators binds intracellular calcium with increasing affinity, providing a sensitivity range to match many applications. Oregon Green probes emit green fluorescence at resting levels of Ca2+ and increase their fluorescence intensity 14-fold with increasing Ca2+ concentration. The cell-permeant formulation (Cat. No. O6807) can be loaded in cell media and is compatible with flow cytometry.
-Rhodamine-based calcium indicators comprise a range of probes for large or small changes in Ca2+ concentration. They exhibit a 50-fold increase in fluorescence upon calcium binding and offer a range of wavelengths that can be used in conjunction with GFP or green-fluorescent dyes for multiplexing. Rhod-2, AM (Cat. No. R1245MP), in particular, localizes to mitochondria and can be used with flow cytometry.
-Membrane potential: A distinctive feature of the early stages of apoptosis is the disruption of the mitochondria, including changes in membrane and redox potential. We offer a range of products specifically designed to assay mitochondrial membrane potential in live cells by flow cytometry, with minimal disruption of cellular function. The MitoProbe family of mitochondrial stains (Cat. Nos. M34150, M34151, and M34152) provide quick, easy, and reliable flow cytometric detection of the loss of mitochondrial membrane potential that occurs during apoptosis. MitoTracker dyes (Cat. Nos. M7510 and M7512) are membrane potential-dependent probes for staining mitochondria in live cells. The staining pattern of MitoTracker dyes is retained throughout subsequent flow cytometry immunocytochemistry, DNA end labeling, in situ hybridization, or counterstaining steps. The Mitochondrial Permeability Transition Pore Assay (Cat. No. M34153) provides a more direct method of measuring mitochondrial permeability transition pore opening than assays relying on mitochondrial membrane potential alone. The mitochondrial permeability transition pore (MPTP) is a non-specific channel formed by components from the inner and outer mitochondrial membranes, and appears to be involved in the release of mitochondrial components during cell death.
-Phagocytosis: In phagocytosis, cells internalize particulate matter such as microorganisms, and this process is important for immune responses and during the clearance of apoptotic cells. Probes for studying phagocytosis include BioParticles indicators—bacteria and yeast labeled with fluorescent dyes.
-Tracking phagocytosis using a quench/wash-based assay can report on simple uptake, or a pH indicator can be used to monitor stages in the pathway. We have no-wash assays labeled with pHrodo Red or Green (Cat. Nos. A10010, P35361, P35364, P35365, P35366, and P35367) and no-wash assays for whole blood (Cat. Nos. A10025, A10026, P35381, and P35382), all suitable for flow cytometry.
-pH changes: Sensitive pH determinations can be made in a physiological range using either fluorescent intensity or ratiometric measurements. pHrodo dyes (Cat. Nos. P35373 and P35372) provide signal intensity modulation from pH 2 to pH 9 and with a choice of fluorescent wavelengths. Tracking internalization of fluorescent dextran is a routine method for analyzing pH changes in cellular compartments. Dextran conjugates of pHrodo dyes (Cat. Nos. P35368 and P10361) provide the most complete solution by allowing discrimination of vesicles from early endosomes to lysosomes, with no quench or wash required.
-Reactive oxygen species: Cells that are environmentally stressed usually contain greatly increased levels of reactive oxygen species (ROS). CellROX reagents are fluorogenic probes developed for the detection and quantitation of ROS in live cells. These cell-permeant reagents are non-fluorescent or very weakly fluorescent in the reduced state; however, when oxidized, they become brightly fluorescent and remain localized within the cell. We offer CellROX Green (Cat. No. C10492), CellROX Orange (Cat. No. C10493), and CellROX Deep Red (Cat. No. C10491) Assay Kits validated for flow cytometry.

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The difference between these products is in the final working concentration of the reagent and incubation time. For flow cytometry applications, we recommend using a final concentration in the range of 0.5 to 1 µM with an incubation time ranging from 2 to 3 hrs. For imaging, we recommend a final concentration in the range of 1 to 10 µM with an incubation time ranging from 30 mins to 1 hr. For both applications, one should optimize the final working concentration and incubation time.

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The CellEvent Caspase-3/7 Green Detection Reagent is not covalently attached to any cellular component and may be lost upon fixation. For flow cytometry applications, after applying the reagent, you should examine without washing or fixation. For imaging applications, the CellEvent product signal may be retained after fixation in 3.7% formaldehdye for 15 min.

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The approximate excitation and emission peaks of this product after hydrolysis are 630 nm and 661 nm, respectively. Cells labeled with CellTrace Far Red reagent can be visualized by fluorescence microscopy using standard Cy 5 filter sets or analyzed by flow cytometry in an instrument equipped with a 633/635 nm excitation source and APC channel.

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Monoclonal secondary antibodies are useful in detecting specific isotypes of human, mouse, and rat immunoglobulins. They may be used in ELISA and flow cytometry applications. Ready-made antibody cocktails are also available for measuring different IgG isotypes. We also offer monoclonal antibody products directed to rabbit IgG.

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The “UpCell surface with grid” products have a 3 x 3 mm grid design over the entire culture surface. Cell attachment and growth is restricted to within these small squares, preventing cells from clumping, and enabling easier harvest of the cells as single cells or small cell clusters. Products with “UpCell surface with grid” are suitable for the preparation of cells for flow cytometry analysis but cannot be used for cell-sheet applications. We offer 60 mm and 100 mm dishes with “UpCell surface with grid” whereas the 35 mm dishes contain supportive membrane for harvesting of cell sheets.

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eBioscience Super Bright dyes are a series of patent-pending fluorochromes that are based upon a fluorescent polymer and its tandems. Super Bright dyes have been developed for use in flow cytometry and can be excited by the violet laser (405 nm). The peak emission of each Super Bright dye is indicated by the number in the product description, e.g., "Super Bright 600" has a peak emission of 600 nm.

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Samples can be stored at 4 degrees C for up to 16 hours (i.e., overnight) after fixation with BrdU Staining Buffer (with or without washing in Flow Cytometry Staining Buffer) with no adverse effect on performance.

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