Flow Cytometry Frequently Asked Questions
Q. What are good fluorochrome dyes to use in multicolor staining for flow cytometry?
For detailed information about the absorption and emission wavelengths and intensity of the fluorochrome dyes we offer, see our Best Protocols® Fluorescent Dyes Chart or our Fluorochrome Poster. We are more than happy to help you plan your experiments to match the antibodies with the best fluorochromes. Please contact email@example.com should you require further information.
Q. What is compensation?
Compensation is a technique used to eliminate false signal that results from spectral overlap between fluorescent dyes when used in multicolor staining panels. For example, FITC has a broad emission spectra that spills into the PE detector. As a result, the cytometer will register a false population of PE labeled cells. Corrections must be made to avoid this. For further information on compensation visit this website http://www.drmr.com/compensation/index.html.
Q. What antibodies can I use to label a particular cell type for flow cytometric analysis?
Use our Mouse and Human CD charts to help identify the expression pattern of the different antigens of interest. There you will also find information about staining and links to product information regarding each antibody eBioscience has available.
Q. How do I discriminate between live cells and dead cells when doing a flow cytometry analysis?
Discrimination between viable and non-viable cells can be carried out with the use of the 7-AAD Viability Dye (cat. 00-6993) or Propidium Iodide (PI) Staining Solution (cat. 00-6990). The 7-AAD or PI will mark the non-viable cells by binding to the nuclei of those cells. The nucleic acid of viable cells will not be accessible to the dye and will not be stained. When analyzing the data collected, gate out all cells stained with the viability dye. When staining for intracellular proteins, use the Fixable Viability Dye eFluor 455UV, 450, 520, 660, or 780. For more information, please see our Functional Dyes and Reagents webpage.
Q. What do Monensin and Brefeldin A do?
These are critical chemicals for inclusion in cell cultures during cell activation prior to analysis by flow cytometry for the presence of intracellular chemokines and cytokines. Monensin (cat. 00-4505) and Brefeldin A (cat. 00-4506) are protein transport inhibitors that block secretion of proteins by cells via the golgi apparatus, thereby causing an accumulation of cytokines at the endoplasmic reticulum or Golgi. Cells are often incubated with either of these two chemicals during cell activation in order to promote the accumulation of detectable protein levels within the cell. These are often essential reagents that allow intracellular detection of proteins that would otherwise be too low in abundance to detect. Monensin is known to block the transport from the medial to the trans cisternae of the Golgi stack. Brefeldin A has been reported to block protein transport from the ER. Specific information about the use of Monensin and Brefeldin A can be found on their respective Technical Data Sheets.
Q. Can I vortex my antibody or recombinant protein?
We do not recommend vortexing protein containing solutions (such as recombinant proteins or antibodies) as this could alter the functionality of the product. During shipping, it is possible that liquid is dispersed throughout the tube. Prior to use, quick spin the antibody vial to recover the maximum volume.
Q. Can I vortex samples that have been stained with conjugated antibodies?
Yes, you can but keep it short (pulse vortex), as it is always best to maximize the health of your cells.
Q. Can samples stained with antibodies conjugates to tandem dyes (i.e. APC-eFluor® 780, PE-Cy7, etc.) be fixed and stored?
Yes, samples that are stained with tandem dye conjugated antibodies can be stored in IC Fixation Buffer (cat. 00-8222) (100 µL sample with 100 µL IC Fixation Buffer) or 1-step Fix/Lyse Solution (cat. 00-5333) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically.
Q. I can't run my samples immediately. What do I do to preserve the best staining?
For optimal results, we recommend that you analyze stained cells immediately.
However, if you do need to store your samples that were stained with antibodies conjugated to organic fluorochromes, we recommend you complete your staining protocol and fix your samples with IC Fixation Buffer (cat. 00-8222) (100 µL sample with 100 µL IC Fixation Buffer) or 1-step Fix/Lyse Solution (cat. 00-5333). Cells can be stored in these buffers for up to 3 days in the dark at 4°C.
We have observed minimal impact on brightness or FRET efficiency/compensation when using the IC Fixation (cat. 00-8222) or 1-step Fix/Lyse Solution (cat. 00-5333). Differences in fixation buffer quality can affect fluorochrome brightness or FRET efficiency. Fixation of tandem dyes, such as APC-eFluor 780 and PE-Cy7, does not increase the amount of compensation required from the APC or PE detector, respectively. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically.
Q. Can I use homemade fixation buffers (or other commercially available fixative) on my cells?
No. We do not recommend the use of other fixatives. We validate our studies using eBioscience’s IC Fixation Buffer (cat. 00-8222) and 1-step Fix/Lyse Solution (cat. 00-5333). Other recipes or manufacturer’s products may give inferior results.
Q. I read a paper regarding incompatibility of PE-Cy5.5 conjugates and DEC205+ cells. What alternative options are available?
CD205 has been shown to bind to PE-Cy5.5 antibody and streptavidin conjugates (Part et al. 2012 J. Immunol Methods 384:184-90); therefore, this format should not be used on CD205+ dendritic cells. We recommend alternative formats such as PerCP-Cy5.5 or PerCP-eFluor 710 whenever possible.
Storage and Shelf-life
Q. Can I keep the diluted (1 mM) BrdU at 4°C? Can I refreeze it after use?
The stock (32.5 mM) BrdU can be frozen and thawed up to three times. Furthermore, the stock BrdU can be kept at 4°C for up to one week. However, the diluted (1 mM) BrdU should be used immediately.
Q. Is the BrdU supplied as a sterile formulation for in vivo use?
Yes, the BrdU is supplied as a sterile liquid. The vial should be opened under sterile conditions if it will be used in vivo.
Q. How long can I keep the diluted DNase I at 4°C?
Once diluted to a working concentration, the DNase I can be kept at 4°C for up to 1 week. Longer storage under these conditions will result in a reduction in positive staining.
Q. Can I refreeze the DNase I?
Yes, as supplied, the DNase I can be frozen and thawed up to three times.
BrdU kit protocol
Q. What are the stopping points?
Samples can be stored at 4°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.
Q. Has the kit been tested on proliferating cells of different species?
We have used this kit to detect proliferating human and mouse cells. Other species have yet to be tested but feline, non-human primate and canine should also work.
Q. Can Fixable Viability Dyes be used with the BrdU Staining kits for Flow Cytometry?
Yes, a Fixable Viability Dye can be added to samples after BrdU labeling and before fixation with the BrdU Staining Buffer. See the protocol for additional information.
Q. Can I also stain for intracellular targets such as transcription factors and cytokines using this kit?
Yes, primary antibodies against additional intracellular targets can be added alongside the Anti-BrdU antibody. However, it is critical that you empirically determine whether these additional antibodies are compatible with this staining protocol.
Q. I’ve run out of the BrdU Staining Buffer and/or the Anti-BrdU antibody, can I purchase more? Yes, we the following reagents as stand-alone products.
- BrdU Staining Buffer Set (cat. no. 00-5525)
- Anti-BrdU APC (cat. no. 17-5071)
- Anti-BrdU FITC (cat. no. 11-5071)
- Anti-BrdU eFluor® 450 (cat. no. 48-5071)
- Anti-BrdU PerCP-eFluor® 710 (cat. no. 46-5071)
eFluor® General Questions
Q. What is the difference between eFluor® Organic Dyes and NCeVolve™ QDots®?
The eFluor Organic Dyes (eFluor 450, APC-eFluor 780, PerCP-eFluor 710, eFluor 710) are conventional fluorochromes. In contrast, the eVolve™ line of products are Quantum dots.
Q. Can eFluor Organic fluorochromes and eVolve™ QDots® be used in combination with other organic fluorochromes?
Yes, both the eFluor Organic Dyes and eVolve QDots are fully compatible with conventional fluorochromes and can be used in the same manner as the fluorochromes that they are replacing. We recommend the Flow Cytometry Staining Buffer (cat. 00-4222) as stated in the Staining Cell Surface Antigens for Flow Cytometry Protocol.
Q. What buffers are compatible with the eFluor Organic fluorochromes and eVolve™ QDots?
The eFluor Organic fluorochromes and eVolve™ QDots® can be used with flow staining buffers containing PBS and protein.
Q. Can the eFluor Organic fluorochromes be used for intracellular staining?
Yes, the eFluor Organic fluorochromes can be used for intracellular staining. The eFluor organic fluorochromes maintain bright signal and require minimal changes in compensation when fixed with eBioscience’s IC Fixation Buffer (cat. 00-8222) and Permeabilization Buffer (cat. 00-8333) or 1-step Fix/Lyse Solution (cat. 00-5333) (as compared to live cells).
Q. I am unable to analyze my cells stained with eFluor Organic Dyes today. What options do I have?
Your options will depend on the samples you are analyzing.
If cell viability is not critical, you can store your stained samples at 4°C or on ice overnight in the dark and analyze the following day.
For samples stained with eFluor organic fluorochromes, we recommend that cells be suspended in 100 uL of Flow Cytometry Staining Buffer (cat. 00-4222) and 100 uL of eBioscience IC Fixation Buffer (cat. 00-8222); samples can be incubated for up to 3 days at 4°C in the dark. Alternatively, the 1-step Fix/Lyse Solution (cat. 00-5333) can be used. This is a great option when working with whole blood but also works for other cell types.
eFluor® Organic Dyes
Q. Why are you replacing APC-Cy7 and APC-Alexa Fluor® 750 with APC-eFluor® 780, Pacific Blue® with eFluor® 450 and Alexa Fluor® 647 with eFluor® 660?
Due to the dimness, compensation and photostability issues associated with APC-Cy7, we are replacing this fluorochrome with the APC-eFluor 780 tandem dye. This newly released tandem dye will also be replacing the APC-Alexa Fluor 750 fluorochrome. The APC-eFluor 780 tandem dye stains with a similar or brighter staining intensity as APC-Cy7 and APC-Alexa Fluor 750.
Similarly, the eFluor 450 will replace our Pacific Blue® fluorochrome offering and eFluor 660 will replace our Alexa Fluor® 647 offering.
Q. Can the eFluor Organic Dyes be frozen?
As with other organic fluorochromes, we do not recommend the eFluor Organic Dyes be frozen.
Q. Are the eFluor Organic Dyes photo-labile?
As with other fluorochromes, we recommend minimal exposure to light to maintain optimal signal.
Q. What is the excitation/emission wavelength for the eFluor Organic Dyes?
As with all eFluor products, the eFluor Organic Dyes are named for their emission wavelength. For information regarding the peak excitation and emission wavelengths for the eFluor Organic Dyes, please see our Fluorescent Dyes for Flow Cytometric Analysis.
Q. Is the eFluor 660 fluorochrome compatible with Anti-Cy5/Alexa Fluor 647 beads?
Yes, in house studies have demonstrated that the eFluor 660 fluorochrome is recognized by Anti-Cy5/Alexa Fluor 647 beads. Side by side studies with Alexa Fluor 647 versus eFluor 660 conjugated antibodies have demonstrated comparable results.
Q. What are eVolve fluorophores?
eVolve fluorophores were developed as the next logical step after the nanocrystal. eVolves combine the excellent optical properties of QDot® nanocrystals with the conjugation expertise of eBioscience to generate a conjugate without the limitations of previous nanocrystal technologies.
Q. Are eVolve fluorophores the same as eFluor® nanocrystals?
No. While some of the methods to generate eVolve dyes were adapted from nanocrystals, eVolve dyes are unique and free of many of the limitations of previous nanocrystal technologies.
Q. What is the best application for eVolve fluorophores?
eVolve fluorophores are very bright with narrow emission peaks and, as such, are a beautiful addition to almost any multicolor flow cytometry panel. They are not, however, suitable for IHC applications.
Q. Which channels will require compensation with eVolve 605- and 655-conjugated antibodies?
In general, the emission spectra of eVolve-conjugated antibodies are narrow, resulting in minimal spectral overlap with other detectors and thus requiring minimal compensation. The filters and instrument settings used in other detectors will determine the amount of compensation that may be necessary. Please be aware that eVolve 655 will require compensation out of the APC channel.
Q. How does the protocol for staining with eVolve-conjugated antibodies differ from staining with conventional organic fluorochromes or eFluor® NC-conjugated antibodies?
The eVolve-conjugated antibodies may be used in the same fashion as other fluorochromes with routine staining in PBS-based buffers. Antibodies conjugated to eVolves are pre-titrated and diluted to the optimal concentration for ease of use. Please refer to the product Technical Data Sheet.
Q. Which laser do I use to detect eVolve conjugated antibodies?
eVolve-conjugated antibodies are excited by the 355 nm (UV), 405 nm (Violet) and to a lesser extent the 488 nm, 532 nm, and 561 nm lasers. We recommend using the 355 nm or 405 nm laser for optimal performance.
Q. Which filter sets do I need to detect eVolve-conjugated antibodies and where do I place the filters?
|Fluorochrome||Dichroic Pass Filter||Bandpass Filter||Alternative Bandpass Filters|
|eVolve™ 605||595 LP||605/40||605/50, 605/20, 610/20|
|eVolve™ 655||630 LP||660/40|
Placement of filter sets will depend on your current instrument/filter configuration. Please contact eBioscience Technical Support for further details.
Q. Where can I purchase specific filter sets suitable for use with eVolve-conjugated antibodies?
Filter sets can be purchased from Chroma Technology, Omega Optical, or Semrock, Inc.
Q. Can eVolve-conjugated antibodies be used in combination with intracellular staining?
Yes! eVolve conjugates were designed to be compatible with eBioscience’s intracellular staining buffers including the Foxp3/Transcription Factor Staining Buffer Set (cat. 00-5523) and the Intracellular Fixation and Permeabilization Buffer Set (cat. 88-8824).
Q. Will eVolve fluorophores survive fixation?
Absolutely. eVolve fluorophores retain their fluorescent properties after fixation with eBioscience’s fixation buffers such as IC Fixation Buffer (cat. 00-8222).
Q. Can I use homemade fixation buffers (or other commercially available fixatives) on my cells after staining with eVolve-conjugated antibodies?
No. For optimal results, we recommend that you use the IC Fixation Buffer (cat. 00-8222) or 1-step Fix/Lyse Solution (cat. 00-5333). Impurities in fixation buffers can severely affect fluorochrome brightness.
Q. When designing a multi-color panel for flow, what I can do to minimize issues with compensation?
When there is significant spectral overlap between two fluorochromes, it is recommended that the fluorochromes are conjugated to antibodies against markers that stain independent cell populations to obtain optimal resolution. For more information regarding compensation, please see the Compensation Overview page here. Please contact Technical Support firstname.lastname@example.org for further details.
Q. Are eVolve dyes toxic?
All eVolve dyes contain trace amounts of the heavy-metal Cadmium (Cd), in addition to Zinc (Zn) and Selenium (Se). Cadmium is potentially harmful and should be handled with gloves. We recommend that eVolve dyes be handled in accordance with laboratory practices for heavy metals and that users wear appropriate personal protective equipment at all times. These low levels of Cadmium are not likely to pose health risks, however prolonged exposure studies have not been conducted. For further information, please contact Technical Support, email@example.com, to request the Material Safety Data Sheet (MSDS) for these products.
Q. How do I dispose of eVolve conjugates?
The eVolve fluorophores are nanoparticles composed of a Cadmium core. We recommend their disposal in accordance with your local, state, and federal regulations for reagents containing heavy metals. Please contact your institution’s health and safety officers in order to ascertain the proper disposal of this material. For further information, please contact Technical Support, firstname.lastname@example.org, to request the Material Safety Data Sheet (MSDS) for these products.
General OneComp eBead Questions
Q. What host species of antibodies do OneComp eBeads work with?
OneComp eBeads capture mouse, rat and hamster (Armenian and Syrian) antibodies of IgG and IgM classes, independent of light chain. This means OneComp eBeads are compatible with almost all direct-conjugates used in flow cytometry.
Q. Do OneComp eBeads work with antibodies of rabbit origin?
While OneComp eBeads were not designed with this intent, they do exhibit some reactivity to rabbit IgG. In some cases, OneComp eBeads may be useful for compensation of rabbit antibody conjugates, but this must be determined for each antibody.
Q. Can I use OneComp eBeads to compensate for Fixable Viability Dyes or Cell Proliferation Dyes?
No. Since these dyes are not antibodies, they are not compatible with OneComp eBeads. However, it is possible to use cells for this compensation control while using OneComp eBeads for the rest of the normal antibody-stain compensation controls.
Q. Can OneComp eBeads be used with violet laser-excited fluorochromes?
Due to background autofluorescence issues, OneComp eBeads are not optimized for use with violet laser-excited fluorochromes. However, OneComp eBeads can be used with eFluor 450- and Pacific Blue-conjugated antibodies because there is no spillover into the closest detector (FITC) to compensate.
Q. Are OneComp eBeads compatible with auto-compensation software?
Yes. After staining, OneComp eBeads provide positive and negative peaks that can be used with auto-compensation software.
OneComp eBead Protocol Questions
Q. Is there flexibility in staining incubation times with OneComp eBeads?
Incubations of 5-90 minutes have been tested and may be suitable for most clones. However, deviation from the suggested 15-30 minute incubation as written in the protocol should be tested on an individual basis.
Q. Is it necessary to wash OneComp eBeads after staining?
One wash with 2 mL of Flow Cytometry Staining Buffer (cat. 00-4222) is recommended for ideal staining of OneComp eBeads. However, it may be possible in some cases to use unwashed OneComp eBeads, if the antibody staining concentration is low enough. In testing, it appears that concentrations starting below 0.06 ug per test may provide appropriate compensation values. Usage in this way should be confirmed in the individual antibody of interest.
Q. Is it necessary to use exactly one test of antibody when staining OneComp eBeads?
No. One test of antibody is recommended for ease of use, since it is the same quantity that would be added to cells in the experiment. However, since the brightness of the stained bead is dependent on the bead’s specificity and not on the test antibody’s specificity, it is not critical that the antibody be used at its optimal concentration. In general, antibodies tested well between 1.0 ug and 0.03 ug per sample. Some antibodies may need titration for optimized performance with OneComp eBeads.
Q. Should fluorescence PMT voltages be set with OneComp eBeads or cells?
Cells. Fluorescence-PMT voltages should be adjusted as desired for unstained cells. Stained cells and stained beads should then be checked to be sure that the voltage settings keep the positive populations on scale. If they are off scale, voltages should be adjusted until the positive populations are on scale. Once you have begun setting up compensation, only forward scatter and side scatter parameters should be adjusted to properly display beads or cells. Changes to any other voltages after compensation setup has begun will result in incorrect compensation settings.
Q. Can one sample tube of OneComp eBeads be used with multiple antibodies at once?
No. OneComp eBeads are designed to be used as single-color compensation controls only.
Optimization of OneComp eBeads
Q. Why do IgM antibodies stain OneComp eBeads less brightly than IgGs and will this affect my compensation values?
IgM antibodies are pentamers in solution. It is believed that these structures cause some steric hindrance resulting in less fluorochrome conjugates being captured by the beads. Compensation values should still be set appropriately using OneComp eBeads.
Q. My antibody seems to stain the beads dimly. Are there any recommendations on how to improve the signal?
Rarely, there may be clones that are not captured well by OneComp eBeads. In such cases, it may be possible to improve the signal brightness by increasing the staining concentration, the incubation time, or both. In some cases, a different antibody that is conjugated to the same fluorochrome may be used instead.
Q. Sometimes my positive population appears as a tight doublet. What should I define or gate as positive?
There is some antibody-based variation on the shape of the positive peak. When the positive peak appears as a doublet, it is appropriate to gate on the entire positive population. Appropriate compensation values will result from this strategy.
Q. OneComp eBeads are a mixture of negative and positive beads. Can I use a universal negative in auto-compensation software?
Yes. Since OneComp eBeads are a pre-mixed suspension of both negative and positive beads, there will always be a tube-specific negative population, but it is not required that the software use this population. Just include an unstained sample along with your single-color controls and be sure the gate for positive events is assigned to the positive population for each sample. The software will then use the universal negative sample and ignore the tube-specific negative populations. This should be true for any analysis software with auto-compensation features, including Flowjo and FACSDiva.