Flow Cytometry Support—Getting Started

Cytometry is the measurement of physical or chemical characteristics of cells or particles. Flow cytometry measures these characteristics of cells or particles as they individually pass lasers in a flow cytometer instrument. Flow cytometry is performed on single cells, providing discrete measurements for each cell in the sample. It also provides a statistical distribution of the measured characteristics of the sample.

A flow cytometer is made up of three subsystems: fluidics, optics, and electronics. Fluidics moves the cells and introduces them for interrogation. Optics generates and collects the light signals. Electronics converts the optical signals to proportional electronic signals for computer analysis.

There are several applications, some of which include immunophenotyping, cell cycle analysis, apoptosis assays such as annexin V staining, CellEvent™ Caspase-3/7 assay, and TUNEL assay, cell viability, proliferation assays such as CellTrace™ assay and Click-iT™ EdU assay, measurements of mitochondrial potential with MitoProbe™ assays, and cell counting using counting beads.

• Measures data from single cells.
• Data are obtained for a large number of cells, generating a rich statistical analysis of cell populations.
• Because single cells are measured, it will reveal heterogeneity within a population.
• With the ability to multiplex, small sub-populations can be identified.
• Thousands of cells can be analyzed rapidly.
• It is ideally suited for blood samples and other cells in suspension.
• Data can be re-analyzed multiple times after acquisition.
• Flow cytometry files (FCS) can be archived.

With the option to be configured with up to 4 lasers and 14 colors for multi-parameter analysis the Attune™ NxT Acoustic Focusing Cytometer was designed as a modular system to fit most experimental needs and lab budgets. The novel design of the optical path helps ensure precise fixed alignment of four spatially separated lasers onto the sample stream enabling consistency in data over time, superior performance, and superior reliability. The instrument can be configured with up to 4 solid-state lasers (405 nm, 488 nm, 561 nm, and 637 nm) with flat top beam profiles. 

The Attune™ NxT Flow Cytometer’s acoustic focusing uses ultrasonic radiation pressure (>2 MHz) to transport particles into the center of the sample stream. This pre-focused stream is then injected into the sheath stream, which supplies an additional hydrodynamic pressure to the sample. The combination of these two forces—termed acoustic-assisted hydrodynamic focusing—results in a narrow core stream and uniform laser illumination, regardless of the sample input rate. In traditional cytometers that rely solely on hydrodynamic focusing, the sample core widens to accommodate the increases in flow rate, which results in less uniform laser light illumination.

• Modular design—Multiple configurations available—field upgradable.
• Save time—10X faster speeds with no loss in data quality.
• Simplified sample prep—No wash, no lyse options, non-clogging fluidics.
• Enables unique applications—Complex protocols on a broad range of cell types and samples.

The smallest size that you can detect with the Attune™ NxT Acoustic Focusing Cytometer is 0.5 µm.

No. The installation of the Attune Nxt Blocker Bar must be done on site by a Themo Fisher Scientific engineer.

• When you titrate a new antibody, we recommend looking at a range of concentrations above and below the recommended amount, perhaps 0.5–20 µL.
• Plot your data on a histogram, and select the concentration where the separation of the mean fluorescence intensity of the positive and negative populations is the greatest.
• The negative can either be the negative peak if you are looking at a mixed population, or an isotype control.
• If you use too little antibody, the separation is small because the positive population does not get fully labeled. 
• If you use too much antibody, the separation decreases because you start to get non-specific binding which moves the negative population toward the positive population. 
• If the suggested range does not give you one or the other of these extremes, you may need to extend the titration with more or less antibody.

A primary antibody is the antibody that targets your antigen of interest. In flow cytometry, many primary antibodies are directly labeled with a fluorophore. If this is the case, you do not need a secondary antibody. 

Secondary antibodies are antibodies that bind to your primary antibody. If your primary antibody is raised in mouse, its isotype is mouse IgG. You then need an antibody that targets mouse IgG. This can be raised in any animal – goat and donkey are commonly used.

No, if your primary antibody is directly conjugated with a fluorophore then you do not need a secondary. You only need a secondary antibody if your primary antibody is unconjugated.

If you are using more than one primary antibody, they need to be from different species, such as mouse and rabbit. If they are from the same species, your secondary antibody will label both primary antibodies. Your secondary antibodies need to be raised in a species other than the ones your primary antibodies are raised in. For example, if you have rabbit anti-target and mouse anti-target primary antibodies, you cannot use a rabbit anti-mouse secondary. This is because when you come in with your anti-rabbit secondary, it will label the rabbit anti-mouse secondary as well. There is no problem with using the same host for all of your secondary antibodies. In this example, use of goat anti-mouse and goat anti-rabbit secondary antibodies would be a good choice.

Yes. The FIX & PERM™ Cell Permeabilization Kit is used in flow cytometry for fixing and permeabilizing cells in suspension, allowing access of antibody to intracellular targets without sacrificing scatter characteristics. It has been validated for use in cell lines, whole blood, and bone marrow. In addition fixing and permeabilizing cells, it lyses red blood cells. It is labeled as a general purpose reagent (GPR) and can be used in clinical settings, but it is also used widely in research labs.

Yes. Here are a couple of references:

Haranaga S, Yamaguchi H, Friedman H et al.(2001) Chlamydia pneumoniae infects and multiplies in lymphocytes In vitro. Infect Immun 69:7753–7759.

Al-Mufti R, Hambley H, Albaiges G et al. (2000) Increased fetal erythroblasts in women who subsequently develop pre-eclampsia. Hum Reprod 15:1624–1628. 

Yes. Generally, you need to increase the incubation time in the permeabilization buffer (Reagent B). Here are a couple of references:

• Millard I, Degrave E, Philippe M et al. (1998) Detection of intracellular antigens by flow cytometry: comparison of two chemical methods and microwave heating. Clin Chem 44:2320–2330.
• Parker JE, Mufti GJ, Rasool F et al. (2000) The role of apoptosis, proliferation, and the Bcl-2–related proteins in the myelodysplastic syndromes and acute myeloid leukemia secondary to MDS. Blood 96:3932–3938.

• Cal-Lyse™ premixed lysis solution (Cat. Nos. GAS010 and GAS010S100) is an IVD reagent specifically formulated to lyse erythrocytes following monoclonal antibody staining of whole blood or bone marrow. Treatment with this reagent leads to both the lysis of red blood cells and the fixation of white cells. Treatment does not affect fluorophore-labeled antibodies bound to leukocytes, and leaves morphological scatter characteristics of leukocytes intact.
• High-Yield Lyse (Cat. No. HYL250) is a premixed, fixative-free erythrocyte-lysis solution that can be used to help eliminate red cells from whole blood for flow cytometric analysis with minimal loss of rare blood cell populations. Using this reagent, lysis of erythrocytes is performed immediately following staining of the blood samples with fluorophore-conjugated monoclonal antibodies; there is no need for a wash step.

TransFix™ Cellular Antigen Stabilizing Reagent (Cat. Nos. FIX2, FIX20, and FIX100) is a whole blood preservative used for the stabilization of leukocytes and the preservation of antigenic sites for future cellular analysis. It enables simple batch-processing of multiple samples for optimal laboratory workflow. Additionally, blood that has been stabilized with TransFix™ reagent can be collected and transported to other sites for analysis. It is easy to use, cost-effective, proven in multiple publications, convenient, and flexible.

People frequently use serum from the same species as their fluorescent antibody for blocking. This is usually the primary antibody if no secondary antibody is used, but would be the host of the secondary antibody if secondary antibodies are used. An alternative to mouse serum, for use with directly labeled mouse primary antibodies, is Fc block. CD16 + CD32 Antibody (FRC-4G8) (Cat. No. MFCR004) is a low-affinity receptor for the Fc region of immunoglobulin gamma complexes.

Alignflow™ Flow Cytometry Alignment Beads are reliable references for aligning, focusing, and calibrating flow cytometers. The Alignflow™ beads are highly uniform with respect to size and fluorescence intensity, resulting in low CVs for both FSC and intensity. The beads approximate the size, emission wavelength, and intensity of many biological samples and permit the calibration of the flow cytometer’s laser source, optics, and stream flow without wasting valuable and sensitive experimental material. They are available in 2.5 µM and 6.0 µM sizes for blue, red, and UV lasers. Alignflow™ Flow Cytometry Alignment Beads for UV Lasers are optimally excitable between 350–370 nm with emission in the range of 400–470 nm, but they can also be excited by a violet laser.

Cell Sorting Set-up Beads (Cat. Nos. C16506, C16507, C16508, and C16509) are reliable standards for flow cytometry cell sorter instrument setup and calibration. The Cell Sorting Set-up Beads approximate the size, emission wavelength, and intensity of many biological samples, and so can be used to calibrate a flow cytometer’s cell sorting system, including laser source, optics, and stream flow. The beads are optimized for checking cell sorter settings such as drop delay and efficiency (cell loss during sorting). Cell Sorting Set-up Beads are available for use with UV, blue, green/yellow, and red lasers.

Our Flow Cytometry Size Calibration Kit has non-fluorescent particle-size calibration standards that provide a simple, accurate way to determine cell sizes by flow cytometry. The kit contains six suspensions of highly uniform polystyrene microspheres with the following diameters: 1.0 µm, 2.0 µm, 4.0 µm, 6.0 µm, 10.0 µm and 15.0 µm. All of the microsphere suspensions are provided in convenient dropper vials. This kit looks at the scatter properties of non-fluorescent microspheres of known sizes to determine approximate cell sizes. 

As an alternative, you may covalently attach your antibody to colorless, 5 µm Aldehyde/Sulfate Latex Beads (Cat. No. A37306). The aldehyde moiety on the bead surface directly binds with primary amines on the surface of the antibodies in simple buffers such as PBS (pH 7.4). Make sure that the buffer does not contain any primary amines (i.e., do not use Tris or glycine buffers). To avoid making bead-antibody-bead-antibody multimers, add excess antibody relative to the beads.

Yes, the AbC Total Antibody Compensation Bead Kit can bind Fab dimers as long as the Fab dimers are derived from either mouse, rat, hamster, or rabbit species.

Here are some tips to follow:
- Make sure that the beads were never frozen.
- Avoid mixing by vortexing too long (no longer than 10 seconds) or sonication as this may promote denaturation of the protein on the surface of the beads.
- Use the product within the warranty period (one year).

Yes. The succinimidyl ester (NHS ester) and isothiocyanate (e.g., FITC, TRITC) dye derivatives may be attached to the ArC beads, but you would have to optimize the amount of reactive dye per ArC beads.

This is because succinimidyl ester (NHS ester) groups are very labile to water; they dissociate readily from the dye structure if held in solvents that are contaminated with water, as is possible from condensation or absorbing water from the atmosphere.

This can occur if the solution of beads was ever frozen. The formation of ice excludes the beads and compresses the beads together. Once frozen, the beads cannot be resuspended into solution and are no longer usable.

These are polystyrene beads.

This may be caused by aggregation of the beads. Mix thoroughly before dispensing a drop from the stock vial and mix again after adding buffer. Aggregation may be also be caused by microbial contamination or if the beads were frozen. In these cases, they can no longer be used.

No. You must always compensate with the same fluorophore present on the samples. The GFP BrightComp eBeads Compensation Bead Kit spectrally emulates GFP and must only be used on GFP-expressing samples.

An isotype control is a non-targeted antibody that matches the isotype of the primary antibody. It is used to control for non-specific binding of the primary antibody.

The flow cytometry field is moving away from using isotype controls as they are not necessarily the most appropriate way to control for non-specific binding. Instead, they are using unstained cells to define the negative population, single stained controls to set compensation, and flow minus one (FMO) controls to set regions and gates.

You may want to consider whether using an isotype control is something you need to do. Here are some references you might want to look at:

• O'Gorman MR, Thomas J. (1999) Isotype controls—time to let go? Cytometry 38:78–80. 
• Keeney M, Gratama JW, Chin-Yee IH et al. (1998) Isotype controls in the analysis of lymphocytes and CD34+ stem/progenitor cells by flow cytometry—time to let go! Cytometry 34:280–283. 
• Hulspas R, O'Gorman MR, Wood BL et al. (2009) Considerations for the control of background fluorescence in clinical flow cytometry. Cytometry B Clin Cytom 76:355–364.
• Enumeration of Immunologically Defined Cell Populations by Flow Cytometry; Approved Guideline—Second Edition. Clinical and Laboratory Standards Institute, (CLSI). Document H42-A2 Volume 27 No.16, 2007. 
• Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells; Approved Guideline—Second Edition. Clinical and Laboratory Standards Institute, (CLSI). Document H43-A2 Volume 27 No. 11, 2007. 

An FMO control (flow minus one) is a control in which you label cells or beads with every color in your panel, omitting one. Make one FMO control for each color in your panel. These controls are important for helping you properly set gates on your data.

All that you need for compensation controls is an unstained sample of your cells (or negative control beads) and samples with each of your fluorophores— one per tube. You want the single-stained controls to be as bright as you expect your brightest sample to be. Antibodies can be bound to beads instead of cells using our AbC™ Total Antibody Compensation Bead Kit (Cat. Nos. A10513 and A10497). 

In a perfect world, the fluorescence emission profile for each individual fluorophore would be a very intense, narrow peak, well separated from all other emission peaks. In reality, organic dyes and fluorescent proteins have broad emission peaks, and compensation must be employed (during or after data acquisition) to correctly assign fluorescence signal to each fluorophore. Compensation is important because it removes fluorescent signal from overlapping spectra so you know that the signal you see is only the signal from the fluorophore of interest. 

Yes, you should have a compensation control for each color in your assay. For amine-reactive stains such as our LIVE/DEAD™ Fixable Dead Cell Stain Kits, you can label beads to make compensation controls using our ArC™ Amine Reactive Compensation Bead Kit (Cat. No. A10346). For other stains, such as cell cycle stains, you need to use labeled cells for your compensation control.

Note: This is a generic labeling protocol to use as a guideline for developing your own assay:

1. Adjust cells to 1 x 10E7 cells/mL.
2. Transfer 100 μL to each tube to be labeled.
3. Add an appropriate amount of antibody to the cells
   (If there is no specified amount to use, do a titration curve, i.e., 1.2 μL, 2.5 μL, 5.0 μL, 10 μL, 20 μL and select the amount with the best resolution).
4. Mix gently and incubate for 15–30 mins on ice.
5. Wash: Add 2 mL of PBS+1% BSA, centrifuge, decant.
6. Add 2 mL of PBS+1% BSA.
7. Analyze by flow cytometry.

Cell counting using flow cytometry can be accomplished by adding an internal microsphere counting standard to the flow cytometric sample. The number of reference beads that are collected reflects a known volume. This allows you to calculate cell concentration.

It depends on your instrument. The Attune NxT™Acoustic Focusing Cytometer uses volumetric syringe pumps that enable absolute cell counting without beads. This is in contrast to cytometers that use traditional hydrodynamic focusing to align the sample.

Bead-free cells can be isolated using Dynabeads™ kits that include DETACHaBEAD™ technology. These include the Dynabeads™ FlowComp™ kits and Dynabeads™ CELLection™ Cell Isolation Kits. Also, any of the negative isolation kits will generate cells that are bead-free. 

Human Cells

Dynabeads™ FlowComp™ Human CD4 Kit (T cells) (Cat. No. 11361D)

Dynabeads™ FlowComp™ Human CD8 Kit (T cells) (Cat. No. 11362D)

Dynabeads™ FlowComp™ Human CD3 Kit (T cells) (Cat. No. 11365D)

Dynabeads™ FlowComp™ Human CD14 Kit (monocytes) (Cat. No. 11367D)

Dynabeads™ CD34 Positive Isolation Kit (hematopoietic progenitor cells) (Cat. No. 11301D)

Dynabeads™ CD4 Positive Isolation Kit (T cells) (Cat. No. 11331D)

Dynabeads™ CD8 Positive Isolation Kit (T cells) (Cat. No. 11333D)

Dynabeads™ Regulatory CD4+/CD25+ T Cell Kit (Cat. No. 11363D)

DETACHaBEAD™ CD19 Kit (Cat. No. 12506D)

CELLection™ Epithelial Enrich Dynabeads™ (Cat. No. 16203)

Dynabeads™ Human Dentritic Cells (DC) Enrichment Kit (Cat. No. 11308D)

Mouse Cells

Dynabeads™ FlowComp™ Mouse CD4 Kit (T cells) (Cat. No. 11461D)

Dynabeads™ FlowComp™ Mouse CD8 Kit (T cells) (Cat. No. 11462D)

Dynabeads™ FlowComp™ Mouse CD4+CD25+ Treg Cells Kit (Cat. No. 11463D)

Dynabeads™ FlowComp™ Mouse Pan T (CD90.2) Kit (Cat. No. 11465D)

Dynabeads™ Mouse Pan T (Thy1.2) (Cat. No. 11443D)

DETACHaBEAD™ Mouse CD4 Kit (T cells) (Cat. No. 12406D)

It depends on your instrument. People have done 17 colors(Perfetto SP, Chattopadhyay PK, and Roederer M (2004) Seventeen-colour flow cytometry: unraveling the immune system. Nat Rev Immunol 4:648–655.) and more, but it takes a lot of planning and testing to make it work. The Attune NxT™ Acoustic Focusing Cytometer can run up to 14 colors plus forward and side scatter with a system equipped with all four lasers (violet, blue, yellow and red).

For help with designing panels for flow cytometry, see our new Molecular Probes Flow Cytometry Panel Design Tool. The panel design tool can help you choose fluorescent antibody conjugates for your flow cytometry panel in a few easy steps: pick the antibody species reactivity, select up to 14 targets of interest (choices include viability dyes), and choose the lasers or fluorophores you want to view.

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

ApoptosisAssays:
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. 

• 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.

Yes, you can. We offer:

• Counting assays: Bacteria Counting Kit, for flow cytometry (Cat. No. B7277) or LIVE/DEAD™ BacLight™ Bacterial Viability and Counting Kit, for flow cytometry (Cat. No. L34856)
• Viability/vitality assays: LIVE/DEAD™ BacLight™ Bacterial Viability Kit (Cat. No. L13152)
• Membrane potential: BacLight™ Bacterial Membrane Potential Kit (Cat. No. B34950)
• Yeast viability/vitality assays: LIVE/DEAD™ FungaLight™ Yeast Viability Kit, for flow cytometry (Cat. No. L34952), FungaLight™ Yeast CFDA, AM/Propidium Iodide Vitality Kit (Cat. No. F34953)

With excitation and emission maxima of 630/650 nm, any standard APC channel may be used. The reagent is efficiently excited using a 633 or 635 nm laser.

A single broad peak may be observed if the dye concentration used is too high or if the dye incubation period is too long. We recommend reducing the amount of dye used and/or the incubation period.

Yes. CellTrace Far Red Dye should always be applied to cell samples first.

Using either a 532 or 562 nm laser, CellTrace Yellow Dye may be detected in the PE/PI emission channel using an Attune NxT Acoustic Focusing Cytometer with 561 nm excitation and a 585/16 nm bandpass emission filter.

We recommend using the CellTrace Yellow Dye if you have either a 532 or 561 nm laser available on your flow cytometer and you want to use the other lasers and channels for other commonly used fluorophores.

A single broad peak may be observed if the dye concentration used is too high or if the dye incubation period is too long. We recommend reducing the amount of dye used and/or the incubation period.

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.

Yes. You can apply surface labels or other reagents. We recommend applying the surface labels or other reagents first.

Yes, you may use SYTOX AADvanced Dead Cell Stain that is included in the CellEvent Caspase-3/7 Green Ready Flow Reagent (Cat. No. R37167) and in the CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit (Cat. No. C10427, C10740). You may also try using other nucleic acid stains in the red or far red channels for staining cells.

No, it must be applied to live cell populations.

We do not recommend doing this. The signal provided with the imaging kits may be too high for use on a flow cytometer.

The middle peak would most likely be from EdU-incorporated cells that have divided, each daughter cell receiving roughly half the incorporated EdU. To avoid this, we recommend shortening the incubation time of EdU with the cells.

The dyes provided in the CellTrace Proliferation kits are amine-reactive dyes that are considered general cytoplasmic stains. They may bind to various membrane proteins, organelles as well as components in the cytoplasm. They are not known to localize in any specific organelles.

A single broad peak is usually caused by using too high a concentration of dye and/or too long an incubation time.

We have not tested the use of the CellTrace reagents for co-culture applications. In theory, this may work, but you would have to test this on your cells of interest.

This is not recommended. The ReadyProbes reagents were developed for imaging applications whereas the Ready Flow reagents were optimized for flow cytometry.

The amount of dye or reagent in the Ready Flow products is proprietary. If you need to consider the exact amount of dye or reagent for your experiment, each of the Ready Flow reagents is available as a standalone product.

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.

Yes, as long as the fluorescent protein does not emit in the green range (˜510–535 nm). For cells that do not express a fluorescent protein, we recommend viewing unstained cells under the FITC channel to examine autofluorescence. For cells expressing a fluorescent protein, we recommend analyzing unstained cell samples to determine the extent the fluorescent protein emission may overlap in the FITC channel.

The Image-iT TMRM Reagent is provided as a 100 µM solution of TMRM in DMSO (5 vials, 100 µL in each vial). The MitoProbe TMRM Assay Kit for Flow Cytometry provides the TMRM as a 20 µM stock solution in DMSO (1 vial, 100 µL) and a vial of CCCP. Both kits have the stock solutions as a 1000X concentration, but final working concentrations required for imaging applications are 5-fold higher than what is needed for flow cytometry analysis.

Yes. The CCCP should be first added to the cell sample, incubated for 5 mins, and then the TMRM should be added and incubated for another 30 mins.

Yes, you may scale up or down as needed, but we recommend keeping the cell density the same. There are 41 µL per drop, so 2 drops is 82 µL (for the 1 mL of sample). You need to only scale down for this 100 µL volume, which would be 8.2 µL.