The following are frequently asked questions about our products and services. If you have a question that is not answered on this page, or if you want clarification, please feel free to contact us. Our customer service and technical support teams are always glad to help.
Call Toll free in the US 888-810-6168 or 858-642-2058 or email us at tech@eBioscience.com.
The Frequently Asked Questions are organized into the following categories:
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Antibody Line
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Recombinant Cytokine Product Line
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Cytokine ELISA Product Line: Antibody Pairs and Ready-SET-Go!
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Cytokine Antibody Pairs Product Line: ELISPOT Applications
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Cytokine Flow Cytometry (Intracellular Staining) Product Line
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Immunohistochemical Staining for Cytokines
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eFluorTM Nanocrystals
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eFluorTM Organic Dyes
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Flow Cytometry / Fluorochrome Dyes
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Functional Assays
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Immunohistochemistry
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Immunoprecipitation
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Western Blotting
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Ordering and Delivery
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Custom Products
Q: What antibodies do you offer?
A: We offer monoclonal and polyclonal antibodies for mouse, human, non-human primate and rat CDs and other cell surface antigens, cytokines and chemokines, apoptosis related antigens and transcription factors. For cross-reactivity, please see our Cross-Reactivity Chart.
Q: What formats do you offer?
A: For convenience, our antibodies are offered in a number of formats. We carry purified and biotin with and without azide, and direct fluorochrome conjugates such as FITC, PE, APC, tandem dyes and an array of Alexa Fluor® conjugates. eBioscience also offers the eFluor™ brand of fluorochromes consisting of both nanocrystal-based and organic fluorochromes.
Q: Do your products come azide-free?
A: Yes, they do. eBioscience's Functional Grade (FG) purified and biotin antibodies are free of preservatives such as sodium azide, and are commonly used in bioassays and in vivo studies.
Q: What is the difference between Purified and Functional Grade Purified?
A: The Purified format of our mAb clones contains sodium azide to maintain sterility of the sample. The Purified product can be used in WB, IP, IHC, and flow. Functional Grade Purified (FG) products contain no preservatives such as sodium azide, and are commonly used in bioassays and in vivo studies. FG products are tested for sterility and meet the industry's standard endotoxin levels of less than 0.001 ng endotoxin/µg antibody; however, the endotoxin levels are often much, much lower than these standards. Because these products contain no preservatives, appropriate measures must be taken to maintain their sterility.
Q: Which fluorochromes do you offer?
A: We carry FITC, PE, PE-Cy5, PE-Cy5.5, PE-Cy7, APC, PerCP-Cy5.5 and Cy5 conjugated antibodies. eBioscience has also introduced the eFluor™ brand of fluorochromes which includes the nanocrystal-based fluorochromes eFluor™ 605NC, eFluor™ 625NC and eFluor™ 650NC and the organic fluorochromes eFluor™ 450 (Pacific Blue® replacement) and APC-eFluor™ 780 (APC-Alexa Fluor 750 and APC-Cy7 replacement). Additionally we offer Pacific Blue®, Alexa Fluor® 488, 647, 700, APC-Alexa Fluor® 750 and Pacific Blue®.
Q: What are the concentrations of your antibodies?
A: Purified, biotin, Functional Grade biotin, FITC and Alexa Fluor® 488 antibodies come in 0.5 mg/ml. APC, PE, Alexa Fluor® 647, 700 and tandem dyes are at 0.2 mg/ml. FG purified antibodies are at 1 mg/ml. These concentrations pertain to all antibodies sold as the ug size. Concentration of test sizes (for many of the anti-human antibodies) are indicated on the Data Sheet in the Applications Tested section.
Q: What is the shelf life of your products?
A: Most of our products have a guaranteed shelf life of one year unless indicated on our Data Sheets. This is provided that they are kept in optimal storage conditions.
Q: Which applications can your products be used for?
A: Our products can be utilized in the following applications:
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Flow cytometric analysis-antigen surface staining (FC)
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Flow cytometric analysis-intracellular staining (ICFC)
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Immunohistochemistry (IHC)
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Immunohistochemistry of frozen sections (IHC/F)
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Immunoblotting (WB)
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Immunoprecipitation (IP)
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ELISA
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ELISPOT
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In vivo assays in mice
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Bioassays
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Blocking/neutralization studies (NU)
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In vitro Cytokine Capture Assay (IVCCA)
Q: Should I order your products per test or per microgram?
A: Most of our products are sold according to mass (per µg), but some are also sold per test.
When you buy a product per test, the product you receive is pretitrated for optimal signal to noise in flow cytometric analysis. No need to spend time finding the optimal dilution of the antibody for your flow experiment, because we've done that for you! Those buying "per test" want a quick and convenient way to get optimal results in their cytometry assays.
The products sold per µg are cheaper, but we don't pretitrate the antibody. Finding the optimal dilution of antibody for your application is up to you. This, however, allows you freedom to use the contents of the vial for various applications at different concentrations.
Q: What information do you have about the cross-reactivity of your antibodies?
A: Information about known cross-reactivity of an antibody is given on the Data Sheets of the antibodies. A good way to check for the absence of cross-reactivity of an antibody with homologous antigens in other species is to look at the isotype of the antibody. The isotype indicates the species in which the antibody was developed. If the anti-rat antibody isotype is "mouse", the antibody probably will not react with mouse cells, because antibodies generally do not recognize antigens in the species they are developed in, unless developed in knock-out mouse. We have done tests to analyze cross-reactivity of anti-human antibodies amongst non-human primates. The results of these tests are summarized in our Cross-Reactivity Charts.
Q: What is the concentration of endotoxin in your Functional Grade (FG) products?
A: FG products are tested for sterility and meet the industry's standard endotoxin levels of less than 0.0001 ng endotoxin/µg antibody for the purified antibodies and less than 0.05 ng/ml for FG biotin antibodies; however, the endotoxin levels are often much lower than these standards. We can provide lot-specific values for the level of endotoxin present in our FG products if you wish.
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Q: What is a ‘unit’ of cytokine activity?
A: A ‘unit’ is defined as the concentration of cytokine required to induce half maximal activity (e.g., in ng/ml). This is also referred to as the 50% effective dose (ED50). This method of expressing potency should only be used for cytokines whose dose-response curves are sigmoidal in shape (e.g., not chemotaxis assays).
Q: What is the relationship between specific activity (Units/mg) and ED50?
A: The formula for converting the activity as an ED50 in ng/ml to specific activity in units/mg is: Specific activity (units/mg) = 10E6 / ED50 (ng/ml)
Q: What is the concentration and specific activity of eBioscience's recombinant protein?
A: Information about our recombinant proteins can be found in the Protein Chart on our Bioassay protocol page. There, specific activity (Units/mg) of the proteins are shown. Some proteins we sell are lyophilized, and need to be reconstituted in buffer. Directions for reconstitution of the protein come with the product and must be followed carefully to ensure proper reconstitution.
Q: What is the difference (or relationship) between laboratory (observed) unitage and international units (e.g., defined by the WHO/National Institute for Biological Standards and Control – NIBSC)?
A: ‘Laboratory’ units are the actual values obtained from running an assay with a particular cytokine in an assay in your lab; i.e., the activity (ED50) you observe on your target cells. ‘International’ units are consensus values of potency derived from a collaborative NIBSC effort to standardize reported use of cytokines. These values are derived from bioassay testing of the same cytokine by many target cell types/substrains. It is very likely that the ‘laboratory’ units you observe and the NIBSC values will not correlate 1:1; e.g., it might take 0.1 - 20 U/ml to see 1 U/ml in your experiment. These are bioassay standards describing potency in bioassay only. The mass values assigned to these are not hard values and use of these for immunoassay standardization is of limited value unless assays calibrated by the NIBSC standard use the same capture and detection antibody clones.
Q: What are the carrier free formats listed for recombinant proteins?
A: These are recombinant proteins that are sterile in 10 mM sodium phosphate, pH 7.2, 150 mM NaCl, with no additional proteins or preservatives. 0.22 µm filtered.
Q. Why can't I see the protein pellet in the vial?
A: eBioscience supplies cytokines in either of 2 forms: as a frozen liquid or lyophilized powder with no carrier protein. The lyophilized cytokines often contain such small amounts of protein that in the lyophilization process these can be deposited on the vial as a thin and, often, invisible film. Before opening, we recommend centrifuging each vial in a microcentrifuge for 20-30 seconds to drive any protein that may be lodged in the cap or on the side to the bottom of the vial. Our quality control procedures ensure that each vial contains the correct amount of product.
Q. Cytokines can be very labile. How should I handle cytokine proteins for greatest stability / shelflife?
A: Follow the specific instructions on each product datasheet. However, in general, the key considerations are the following: 1) always quick-spin vials prior to opening, 2) rehydrate in water (or recommended buffer) to 0.1 ug/ul; invert for 5 minutes at RT, quick-spin, 3) dilute further (ONLY) in a buffered saline which contains 1.0% BSA or 10% FBS as a protein carrier/stabilizer, 4) maintain the master stock at cytokine concentrations of at least 10 ug/ml (long term), 5) make aliquots to minimize the number of freeze/thaw cycles. If the aliquots are frozen, avoid freezers with thermal cycling (e.g., frost-free).
Q: Which cytokines show cross-species activity?
A: With a few exceptions, most human cytokines are active on mouse cells. Many mouse cytokines are active on human cells, but may show lower specific activity than the corresponding human cytokine. A few human cytokines, such as IL-7, even exhibit higher specific activities on mouse cells than do the corresponding mouse cytokines. The interferons, GM-CSF, IL-3, and IL-4 are known to be species-specific with very little, if any, activity on non-homologous cells. In contrast, the FGF's and neurotrophins are very highly conserved and show excellent activity on cells of other animal species.
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Q: Which antibody pair should I use to assay my particular protein in ELISA?
A: Please see our Antibody Pairs Chart on the ELISA protocol page for a complete listing of the capture and detection antibodies, and recombinant protein standards of human, rat, and mouse cytokines.
Q: What other reagents will I need to run an ELISA using your antibody pairs?
A: eBioscience provides all other separate components needed for a successful ELISA. They include the following:
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High-affinity binding NUNC Maxisorp or Corning Costar 9018 ELISA Plates
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Coating Buffer
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Assay Diluent
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Streptavidin-HRP
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ABTS (substrate)
For ELISA pairs, eBioscience recommends the HRP compatible substrate ABTS (read at 405 nm); for the Ready-SET-Go! reagent sets, TMB substrate (read at 450 nm) is included.
Q: What are the performance differences between ABTS and TMB?
A: TMB is a faster developing, stronger substrate (yielding greater amplification/sensitivity/background); use of stop solution produces a yellow end product read at 450 nm. ABTS is a slower-developing blue/green substrate (read at 405 nm), particularly useful for kinetic studies (multiple readings).
Q: What do your Ready-SET-Go! ELISA sets contain?
A: Our Ready-SET-Go! ELISA sets contain pre-titrated capture antibody, pre-titrated detection antibody, calibrated cytokine standard, pre-titrated detection enzyme, coating buffer, assay diluent, and substrate solution.
Q: Can the Ready-SET-Go ELISA sets be used for serum samples?
A: Each Ready-SET-Go set has been specifically engineered (fragmented to Fab’2) for optimal and accurate measurement of cytokines in plasma, serum, and culture supernatant, without interference by serum bearing heterophilic antibody or rheumatoid factor, which can cause false positives when run in assays using intact (non-fragmented) IgG. So, the reagent sets are suitable for assaying tissue culture supernatants, plasma, serum, cell lysates, and other complex biological fluids.
Q: Do the Ready-SET-Go! sets come with pre-coated plates?
A: No, the Ready-SET-Go! sets we offer do not come pre-coated. Instructions for coating the plates are included in the Certificate of Analysis.
Q: What is the sensitivity of your Ready-SET-Go! ELISA sets?
A: The ranges of sensitivity for the human, rat, and mouse recombinant protein standards in our ELISA sets are available on the antibody working conditions chart on our Ready-SET-Go BestProtocols page.
Q: What concentrations should I use with the recombinant cytokine standard in the Ready-SET-Go kits?
A: Each Ready-SET-Go ELISA set comes with its own Certificate of Analysis (C of A). The directions on the C of A should be followed closely to ensure proper use. If you have lost your C of A and need an extra copy, please provide us with your set's lot number and we would be glad to fax you one. The top concentration of recombinant cytokine standard is also available on a table in the Ready-SET-Go BestProtocols page.
Q: What is the ‘Femto-HS’ assay?
A: This is a Ready-SET-Go! set incorporating the new mouse IFN-g capture antibody, clone AN-18. The new AN-18 antibody for mouse IFN-g ELISA and ELISPOT capture delivers much greater sensitivity and much stronger signal than the historical XMG1.2 antibody.
Q: What is the most common reason for no signal in ELISA?
A: Mishandling the recombinant cytokine standard; primarily by diluting this in PBS or water without a protein carrier (causing adsorption to the polystyrene or polypropylene walls) or secondarily by reducing amount of carrier protein stabilizer for freeze/thaw. Aliquoting in too small of a volume (e.g., <20 ul) is also suspected to contribute to poor stability for freezing.
Q: What controls should I run if I have no signal in sandwich ELISA?
A: If you see no signal in ELISA, it may be helpful to first troubleshoot the assay by trying a fresh aliquot of cytokine standard. If this also yields no signal, you can ‘work backwards’ by troubleshooting the detecting antibody/SAV-HRP/Substrate by doing the following control: coat biotinylated detecting antibody at 0.5 ug/ml in PBS (no other proteins - no FBS or BSA) to 2 plate microwells (1 hr at RT); wash; incubate with SAV-HRP, 30 min, RT; wash; substrate. This should yield a very strong signal. If this turns out to be negative, then you will know that there is a problem with your SAv-HRP or substrate reagents.
Q: If I want to collect the TC supernatant daily for a time-course study and run all of the samples at once by ELISA, how could I store the sups?
A: The supernatant can be kept at 4C for short term. Make sure to keep it sterile. For longer storage, you may want to freeze, but avoid multiple freeze/thaw cycles.
Q: Do your ELISA sets cross-react with rat cytokines?
A: Our mouse and human ELISA sets typically do not cross-react with rat cytokines (except for IL-1b, TNF-a, and MCP-1). Rat cytokine ELISA reagents are being developed on an on-going basis. Please check with us.
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Q: Do your antibody pairs for ELISA work in ELISPOT as well?
A: Most, but not all, of our ELISA antibody pairs work for ELISPOT. We continue to evaluate and optimize our anti-human and anti-mouse cytokine antibody pairs for ELISPOT applications. Please see our ELISPOT BestProtocols page for a detailed protocol and a reference table with the list of antibody pairs that can be used in ELISPOT.
Q: What are the critical parameters for successful ELISPOT assay?
A: It is critically important to use a high affinity binding PVDF membrane plate, rather than regular ELISA plate or nitrocellulose membrane plate. Additionally, highest affinity antibodies for capture work best in this assay. Empirical optimization of conditions such as cell density, stimulus/mitogen, and kinetics is necessary.
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Q: What do you recommend as a specificity test for intracellular cytokine staining?
A: To confirm specificity of the staining, it is common to pre-block the directly-labelled anti-cytokine antibodies with excess amounts of cytokine protein (ligand blocking control) prior to staining. Alternatively, unlabelled antibody can be used to stain the cells prior to staining with the labelled antibody. Isotype controls are also helpful for monitoring ‘stickiness’ of the fixed, permeabilized cells and degree of background staining.
Q: What do Monensin and Brefeldin A do?
A: These are critical chemicals for inclusion in cell cultures during cell activation prior to analysis by cytokine flow cytometry. Monensin and Brefeldin A are protein transport inhibitors that block secretion of cytokines by cells via the golgi apparatus, thereby causing an accumulation of cytokines at the endoplasmic reticulum. Cells are often incubated with either of these two chemicals during cell activation before intracellular staining in order to induce accumulate a detectable amount of cytokine without releasing it. These are often essential reagents that allow intracellular detection of cytokines that would otherwise be in too low abundance to detect. Specific information about the use of Monensin and Brefeldin A can be found on their respective technical Data Sheets.
Q: I am not seeing any IL-4 expression by intracellular staining. What controls can I use to optimize my staining?
A: We recommend restimulated cell cultures as a positive control for troubleshooting intracellular cytokine staining experiments. The activation procedures are described in our BestProtocols section. Additionally, we sell these stabilized cell controls as Cytokine IC Control Cells.
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Q: I am interested in staining tissue sections for cytokines. Are your antibodies suitable?
A: Though we do not test the reagents for this application, many of the antibody clones we have available were found to be useful for IHC and have been extensively reported in literature. However, these clones were screened on tissue which was fixed with paraformaldehyde, rather than acetone or methanol. As with intracellular staining for flow cytometry, these cells were also permeabilized with the detergent saponin; saponin treatment is mandatory. The utility of these antibodies for staining paraffin-embedded tissues has not been established. Note that the antibody BVD6-24G2 is specifically not recommended for tissue staining.
Q: Which clones are reported in the literature to be useful for IHC?
A: Mouse: IL-2 (JES6-1A12), IL-4 (11B11; note that BVD6-24G2 is specifically not recommended, though it was published by Bogen et al.), IL-5 (TRFK5), IL-6 (MP5-20F3), IL-10 (JES5-16E3), IFN-g (XMG1.2), GM-CSF (MP1-22E9), TNF-a (MP6-XT22). For protocols, please refer to these papers (particularly Immunol. Rev):
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Sander, B. et al. 1993. J. Immunol. Methods 166:201.
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Sander, B. et al. 1991. Immunol. Rev. 119:65.
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Litton, M. et al. 1997. Am. J. Pathol. 150(5):1607.
Human: IL-2 (MQ1-17H12), IL-4 (MP4-25D2), IL-5 (TRFK5), IL-12 (JES3-12G8), TNF-a (MAb11). For protocols, please refer to these papers:
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Andersson, J. et al. 1994. Immunology 82:16.
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Sander, B. et al. 1991. Immunol. Rev. 119:65.
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eFluorTM Nanocrystals General Questions
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eFluorTM Nanocrystals Flow Cytometry Questions
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eFluorTM Nanocrystals Immunofluorescence Questions
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eFluorTM Nanocrystals Imaging Questions
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eFluorTM Nanocrystals Tools for Discovery Questions
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eFluorTM Nanocrystals Toxicity and Disposal
Q: How do eFluorTM 605NC, eFluorTM 625NC and eFluorTM 650NC conjugated antibodies compare to other fluorochrome conjugated antibodies?
A: eFluorTM Nanocrystals offer several distinct advantages:
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Narrow emission spectra results in less spectral overlap; thus, less compensation is required
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Excellent signal to noise ratio
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Superior photostability compared to conventional fluorochromes
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Compatible with other fluorochromes thereby allowing more flexibility in multiparameter flow cytometry
Q: Are there special considerations I need to take into account when using the eFluorTM Nanocrystal conjugated antibodies for flow cytometry?
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Staining buffer - For the brightest staining intensity we recommend the use of the eFluorTM NC Flow Cytometry Staining Buffer (cat. 00-3222) instead of a PBS-based buffer or eBioscience Flow Cytometry Staining Buffer (cat. 00-4222).
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Lysed Whole Blood (LWB) - Depending on the blood collection method used, you may need to modify your staining procedure. Please see the Flow Cytometry FAQ below for details.
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Intracellular staining - Studies are ongoing to determine the compatibility of the eFluorTM Nanocrystal conjugated antibodies with intracellular staining. Please contact eBioscience Technical Support for further details.
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Long term fixation - We do not recommend long-term fixation, but rather we recommend a fixation time of 30 minutes in 2% paraformaldehyde (PFA) in PBS followed by a wash in eFluorTM NC Flow Cytometry Staining Buffer.
Q: What is the shelf life of eFluorTM Nanocrystal conjugated antibodies?
A: The eFluorTM Nanacrystal conjugated antibodies have a guaranteed shelf life of six months upon receipt or as stated on the vial, provided that they are kept at the optimal storage conditions as stated on the product Technical Data Sheet.
Q: How should I store eFluorTM Nanocrystal conjugated antibodies?
A: As with all fluorochrome conjugated antibodies, we recommend storage at 4-8oC in the dark. Do not freeze. We do not recommend storing dilutions of the antibodies for any length of time.
Q: Are eFluorTM Nanocrystal conjugated antibodies temperature sensitive?
A: The antibodies are stable at 4-8oC. Stability studies indicate that they are not affected by short exposures to higher temperatures (<37oC). Freezing is not recommended.
Q: Are eFluorTM Nanocrystal antibodies light sensitive?
A: They do not exhibit light sensitivity. However, when using in combination with other conventional fluorochrome conjugated antibodies, we do recommend that you protect samples from light.
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Q: Which laser do I use to detect eFluorTM Nanocrystal conjugated antibodies?
A: eFluorTM Nanocrystal conjugated antibodies are excited by the 355nm (UV), 405nm (Violet) and to a lesser extent the 488nm (Argon) lasers. We recommend using the 355nm or 405nm laser.
Q: Which filter sets do I need to detect eFluorTM Nanocrystal conjugated antibodies and where do I place the filters?
Table 1: Recommended Filters for Flow Cytometry
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Recommended Filters for Flow Cytometry
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| Fluorochrome |
Dichroic Pass Filter |
Bandpass Filter |
Alternative Bandpass Filters |
| eFluor TM 605NC
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595 LP
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605/40
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605/50, 605/20, 610/20
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| eFluor TM 625NC
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595 LP
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605/50
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605/40, 625/20
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| eFluor TM 650NC
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630 LP
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660/40
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A. 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 eFluorTM Nanocrystal conjugated antibodies?
A: Filter sets can be purchased from Chroma Technology, Omega Optical, or Semrock, Inc.
Q: Which combination of fluorochromes can I co-stain with eFluorTM Nanocrystal conjugated antibodies?
A: Combinations of fluorochromes which can be co-stained will be dependent on the filter setup of your instrument. With the proper filter setup, eFluorTM Nanocrystals are compatible with the following fluorochromes: FITC, Alexa Fluor® 488, PE, PE-Cy5, PE-Cy5.5, PerCP-Cy5.5, PE-Cy7, APC, Alexa Fluor® 647, Alexa Fluor® 700, APC-Alexa Fluor® 750, and Pacific Blue®. The eFluorTM 605NC and eFluorTM 625NC conjugated antibodies cannot be used in combination with the PE-Texas Red fluorochrome as emissions are similar and excitiation does occur off the 488nm laser.
Q: Can I use eFluorTM 605NC and eFluorTM 625NC conjugated antibodies instead of Pacific Orange®?
A: The eFluorTM 605NC and eFluorTM 625NC conjugated antibodies can be used as an additional fluorochrome off the violet laser. Depending on your filter configuration of the cytometer, you may be able to use the same filter as Pacific Orange®. Please refer to question above which indicates appropriate filters to use.
Q: Can I use the eFluorTM 605NC and eFluorTM 625NC conjugated antibodies together in the same sample?
A: The eFluorTM 605NC and eFluorTM 625NC directly conjugated antibodies cannot be used in combination with one another. The emission spectra are close enough to each other that the available filers cannot distinguish between the two.
Q: Which channels will require compensation with eFluorTM Nanocrystal conjugated antibodies?
A: In general, the emission spectra of eFluorTM Nanocrystal conjugated antibodies are narrow, therefore resulting in minimal spillover into other channels and thus requiring minimal compensation. However, the eFluor™ 650NC conjugates do exhibit some spillover into the detectors for PerCP-Cy5.5 and APC and will require compensation for accurate data acquisition.
Q: What are the concentrations of eFluorTM 605NC and eFluorTM 625NC conjugated antibodies?
A: The eFluorTM Nanocrystal conjugated antibodies have been pre-diluted to the optimal titer; and are available in a “test” size format for your convenience. The recommended volumes of the antibody and its corresponding isotype control are indicated on the product Technical Data Sheet in the “Applications Tested” section.
Q: How does the protocol for staining with eFluorTM Nanocrystal conjugated antibodies differ from staining with conventional organic fluorochrome conjugated antibodies?
A: The eFluorTM Nanocrystals conjugated antibodies may be used in the same fashion as other fluorochromes, although we recommend the use of the eFluorTM NC Flow Cytometry Staining Buffer (cat. 00-3222) for optimal performance. eFluorTM NC Flow Cytometry Staining Buffer is fully compatible with other fluorochromes. The antibodies are provided as “test” sizes pre-diluted to the optimal concentrations for ease of use. Please refer to the product Technical Data Sheet.
Q: Which buffer should I use for staining with eFluorTM Nanocrystal conjugated antibodies? Are there differences in staining in eFluorTM NC Flow Cytometry Staining Buffer compared to PBS-based buffers?
A: We have determined that staining of cells in the recommended eFluorTM NC Flow Cytometry Staining Buffer (cat. 00-3222) provides the most consistent results. While we do see staining in PBS based-buffers, we observe slightly brighter signal intensity when using eFluorTM NC Flow Cytometry Staining Buffer.
Q: How do I prepare the eFluorTM NC Flow Cytometry Staining Buffer?
A: eFluorTM NC Flow Cytometry Staining Buffer (cat. 00-3222) is supplied as a 5X concentrated stock solution. The 5X stock solution should be diluted with distilled water to a 1X solution. The eFluorTM NC Flow Cytometry Staining Buffer does not contain a source of protein and therefore, should be supplemented with 3% Fetal Bovine Serum (final concentration) or equivalent protein prior to use. The buffer should be stored at 4oC.
Q: Is staining whole blood samples compatible with eFluorTM Nanocrystal conjugated antibodies?
A: Samples of whole blood collected directly into Na-Citrate buffer can be stained prior to red blood cell (RBC) lysis. However, when collecting blood in the presence of EDTA, RBCs will need to be lysed prior to staining with eFluorTM 605NC or 625NC conjugated antibodies. With either method, it is recommended to wash and resuspend cells in eFluorTM NC Flow Cytometry Staining Buffer (cat. 00-3222).
Q: Can I fix my cells after staining with eFluorTM Nanocrystal conjugated antibodies?
A: Paraformaldehyde (PFA) fixatives have been found to be compatible with eFluorTM Nanocrystal conjugated antibody staining. We have determined that short incubations (30 min or less) of stained cells in 1 or 2% PFA in PBS causes only a minor decrease of the fluorescence signal. Therefore, we recommend a 30 minute fixation followed by a wash step. When optimal staining sensitivity is required, we do not recommend fixing samples.
Q: How long can cells that have been stained with eFluorTM Nanocrystal conjugated antibodies followed by fixation be stored prior to analysis?
A: We have found the fluorescent signal to be quite stable. Cells which were stained, fixed and then washed have been stored in eFluor™ NC Flow Cytometry Staining Buffer at 4oC up to several days without additional loss (from that observed in the fixation process) in fluorescent signal.
Q: I am unable to analyze my cells today. What options do I have?
A: Your options will depend on the samples you are analyzing.
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If cell viability is not critical, you can store your stained samples at 4oC overnight in the dark and analyze tomorrow.
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Fix the samples for 30 minutes in 2% PFA in PBS, wash the cells 1-2X with eFluorTM NC Flow Cytometry Staining Buffer (cat. 00-3222), then store the cells at 4oC in the dark.
Q: Are the eFluorTM Nanocrystal conjugated antibodies compatible with intracellular staining i.e. cytokines or Foxp3?
A: Studies are ongoing to determine the compatibility of eFluorTM Nanocrystal conjugated antibodies with intracellular staining. Please contact eBioscience Technical Support for further details.
Q: Are there other antibody specificities that will be added to the current eFluorTM Nanocrystal product line?
A: Yes, eBioscience is continually adding new specificities to our eFluorTM Nanocrystal product line. We recommend that you check our website frequently for newly released products. You can also contact the eBioscience Technical Support for further details.
Q: Can I conjugate eFluorTM Nanocrystals to an antibody myself?
A: Reactive eFluorTM Nanocrystals, in a variety of emission spectra, with both carboxyl and amine surface chemistries are available for conjugation. Conjugation protocols are available in our Best Protocols and are provided when you order Reactive eFluorTM Nanocrystals. For more details, please see our Tools for Discovery product listing.
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Q: Can the eFluorTM Nanocrystal conjugated antibodies be used in immunofluorescent (IF) microscopy ?
A: eFluorTM Nanocrystal conjugated antibodies have been successfully used in immunofluorescent microscopy on cells as well as frozen and paraffin-embedded tissue. We have specially formulated Streptavidin eFluorTM Nanocrystals for use in this application. Please refer to our Best Protocols for use of eFluorTM Nanocrystals in immunofluorescent microscopy. Investigation is ongoing for their suitability in other cell and tissue formats, in addition to other staining procedures. Please contact eBioscience Technical Support for further information.
Q: Is the use of a Pap pen compatible with eFluorTM Nanocrystal conjugated antibody staining?
A: We have found that a Pap pen will quench Streptavidin eFluorTM Nanocrystal signal, and therefore its use should be avoided.
Q: Are there other antibody specificities, formulated for immunofluorescent microscopy, which will be added to the current eFluorTM Nanocrystal product line?
A: Yes, eBioscience will be adding new specificities for immunofluorescent microscopy. We recommend that you check our website frequently for newly released products. You can also contact eBioscience Technical Support for further details.
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Q: When used for in vivo imaging, are eFluorTM Nanocrystals toxic to a host animal?
A: The eFluorTM 700NC, designed for in vivo imaging, are cadmium-free and consist of a core composed of Indium Gallium Phosphide (InGaP). These particles are non-toxic to the host animal.
Q: Is there a recommended protocol for using eFluorTM Nanocrystals for in vivo imaging?
A: A protocol for using eFluorTM Nanocrystals for in vivo imaging is provided in our Best Protocols section.
Q: What equipment do I need to visualize in vivo imaging with eFluorTM Nanocrystals?
A: Any system with a compatible laser configuration can be used such as the Dual modality Kodak4000IS optical and X-ray imaging system.
Q: Can the eFluorTM Nanocrystals be used for cell labeling?
A: The eFluorTM Nanocrystals can be used for cell labeling experiments. We recommend using the carboxyl or amine Reactive eFluorTM Nanocrystals due to their surface charge, which will enable passage across the cell membrane.
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Q: Which biomolecules can be conjugated to eFluorTM Nanocrystals?
A: eFluorTM Nanocrystals have been successfully conjugated to peptides, proteins and oligonucleotides.
Q: Which protocol do I use for conjugation of a biomolecule to eFluorTM Nanocrystals?
A: Protocols for conjugation of either amine or carboxyl eFluorTM Nanocrystals are provided in our Best Protocols. These protocols will also be provided with your order of Reactive eFluorTM Nanocrystals.
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Q: Are eFluorTM Nanocrystals toxic?
A: All eFluorTM Nanocrystals, except eFluorTM 700NC which has the non-toxic InGaP composition, 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 eFluorTM Nanocrystals be handled in accordance with laboratory practices for heavy metals and 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 view the Material Safety Data Sheet (MSDS) for these products available on our website.
Q: How do I dispose of eFluorTM Nanocrystals?
A: The eFluorTM Nanocrystals, except those with the non-toxic InGaP composition, are particles 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 on the composition of these materials, please view the Material Safety Data Sheet (MSDS) available on our website.
Q: What is the difference between eFluor™ Organic Dyes and eFluor™ Nanocrystals?
A: The eFluor™ Organic Dyes (eFluor™ 450 and APC-eFluor™ 780) are conventional fluorochromes. These organic dyes are not part of the eFluor™ Nanocrystals line of products.
Q: Why are you replacing APC-Cy7 and APC-Alexa Fluor® 750 with APC-eFluor™ 780, and Pacific Blue® with eFluor™ 450?
A: 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.
Q: Can eFluor™ Organic Dyes be used in combination with other organic fluorochromes?
A: Yes, the eFluor™ Organic Dyes are fully compatible with conventional fluorochromes and can be used in the same manner as the dyes that they are replacing.
Q: What buffers are compatible with the eFluor™ Organic Dyes?
A: The eFluor™ Organic Dyes can be used with flow staining buffers that are compatible with conventional fluorochromes.
Q: Can the eFluor™ Organic Dyes be used for intracellular staining?
A: Yes, as with APC-Alexa Fluor® 750 and Pacific Blue® fluorochromes, the eFluor™ Organic Dyes can be used for intracellular staining. As with the dyes they are replacing, we do recommend a minimum fixation time and data collection as soon as possible for optimal signal.
Q: What is the excitation/emission wavelength for the eFluor™ Organic Dyes?
A: The APC-eFluor™ 780 is excited by the 633/635 nm or 647 nm laser lines on the majority of flow cytometers and has an emission of 765 nm - 780 nm. The eFluor™ 450 is excited by the 405 nm laser and emits at 450 nm. As with all eFluor™ products, the eFluor™ Organic Dyes are named for their emission wavelength.
Q:Can the eFluor™ Organic Dyes be frozen?
A: As with other organic fluorochromes, we do not recommend the eFluor™ Organic Dyes be frozen.
Q: Are the eFluor™ Organic Dyes photo-labile?
A: As with other fluorochromes, we recommend minimal exposure to light to maintain optimal signal.
Q: What is the shelf-life of the eFluor™ Organic Dyes?
A: The APC-eFluor™ 780 has a guaranteed shelf-life of 6 months from date of receipt. The eFluor™ 450 has a guaranteed shelf-life of 1 year from date of receipt. This is provided that they are kept in optimal storage conditions as stated on the product Technical Datasheet.
Q: I am unable to analyze my cells stained with eFluor™ Organic Dyes today. What options do I have?
A: If you are unable to analyze your cells immediately, we recommend that you fix your samples for 30 minutes, wash the cells to remove the fixative, and store overnight in flow staining buffer.
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Q: Which fluorochromes do you offer?
A: We carry FITC, PE, PE-Cy5, PE-Cy5.5, PE-Cy7, APC, APC-Cy5.5, APC-Cy7, Cy5, Pacific Blue®, Alexa Fluor® 488, 647, 700, APC-Alexa Fluor® 750, PerCP-Cy5.5, eFluor™ 605NC, eFluor™ 625NC, eFluor™ 450 and APC-eFluor™ 780 conjugated antibodies.
Q: Can I use APC-Alexa Fluor® 750 interchangeably with APC-Cy7?
A: Yes, APC-Alexa Fluor® 750 is equivalent to APC-Cy7. Both tandems are excited with the 633nm laser and emit at ~775nm. APC-Alexa Fluor® 750 is as bright or slightly brighter and more stable than APC-Cy7; it requires similar compensation. For most experiments they can be used interchangeably
Q: What are the concentrations of your antibodies?
A: Purified, biotin, Functional Grade biotin, FITC and Alexa Fluor® 488 antibodies come in 0.5 mg/ml. APC, PE, Alexa Fluor® 647, 700 and tandem dyes are at 0.2 mg/ml. FG purified antibodies are at 1 mg/ml. These concentrations pertain to all antibodies sold as the ug size. Concentration of test sizes (for many of the anti-human antibodies) are indicated on the Data Sheet in the Applications Tested section.
Q: Can fluorochrome conjugated antibodies be used in applications other than flow cytometry?
A: Our fluorochrome conjugated antibodies are developed expressly for flow cytometric analysis. Those who wish to utilize our fluorochrome dyes for other applications such as fluorescence microscopy will have to evaluate the applicability of the fluorochrome conjugates for such assays themselves.
Q: What antibodies can I use to label a particular cell type for flow cytometric analysis?
A: Use our BestPhenotyping Markers chart on the flow cytometry page to identify the antigen expressed only on your cell type of interest. There you will also find the appropriate antibody that recognizes the antigen, 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?
A: Discrimination between viable and non-viable cells can be carried out with the use of the 7-AAD viability dye (eBioscience product 00-6993). Add 5 µl per million cells of this dye in the samples and incubate for 5 minutes before analysis on the flow cytometer. The 7-AAD 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 observed typically in the third channel of the 488nm laser.
Q: Do you carry Cychrome-conjugated antibodies?
A: Yes we do! "Cychrome" is actually a PharMingen product name for the R-Phycoerythrin/CY5 tandem dye. eBioscience has the same product, but to avoid confusion with brand names, we refer to the dye simply as PE-Cy5.
Q: What are good fluorochrome dyes to use in multi-color staining for flow cytometry?
A: PE and FITC and Alexa Fluor® 488 are the commonly used dyes for multiple staining with a 488nm laser. The PE-Cy5 tandem dye is bright, excited at 488nm, and has an emission spectrum clearly distinguishable from PE and FITC, making it a good third dye to use in three color staining with a single laser. Other dyes include APC, APC-Cy7, and PE-Cy7 and the Alexa Fluor® 647 and 700 and Pacific Blue®. Multiple laser flow cytometers are needed in the simultaneous use of these. APC-Alexa Fluor® 750 is equivalent to APC-Cy7. Both tandems are excited with the 633nm laser and emit at ~775nm. APC-Alexa Fluor® 750 is as bright or slightly brighter and more stable than APC-Cy7; it requires similar compensation. For most experiments they can be used interchangeably. For detailed information about the molecular weight and the absorption and emission wavelengths and intensity of these fluorochrome dyes, see our BestProtocols 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 us should you require furthur information.
Q: What is compensation?
A: Compensation is a technique used to compensate for the spectral overlap that may occur between fluorescent dyes during dual antigen staining. For example, FITC emits at wavelengths that are picked up by the PE channel. 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 www.drmr.com.
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Q: Do you have purified antibodies that can be used in vivo in mice?
A. eBioscience's Functional Grade (FG) antibodies are free of preservatives such as sodium azide. This makes the FG format appropriate for bioassays and in vivo studies in mice. In addition, these are tested for sterility and meet the industry's standard endotoxin levels of less than 0.01 ng endotoxin/µg antibody; however, the endotoxin levels are often much lower than these standards. Because these products contain no preservatives, appropriate measures must be taken to maintain their sterility.
Q: What is the difference between Purified and Functional Grade Purified?
A: The Purified format of our mAb clones contain sodium azide to maintain sterility of the sample. The Purified product can be used in WB, IP, IHC, and flow. Functional Grade Purified (FG) products contain no preservatives such as sodium azide, and are commonly used in bioassays and in vivo studies. FG products are tested for sterility and meet the industry's standard endotoxin levels of less than 0.001 ng endotoxin/µg antibody; however, the endotoxin levels are often much lower than these standards. Because these products contain no preservatives, appropriate measures must be taken to maintain their sterility.
Q: What concentration and regimen should I use to inject antibodies for in vivo studies?
A: Our in vivo use BestProtocols page gives a list of antibodies used for in vivo studies and a list of references that report their usage.
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Q: Can I use eBioscience antibodies in immunohistochemical (IHC) assays?
A: If the products have been reported in the literature for use in IHC, it will be so expressed in the product's Technical Data Sheet. eBioscience does quality control testing on selected products using immunohistochemical staining of frozen tissues, immunoprecipitation, and immunoblotting.
Q: Will these antibodies work in Formalin Fixed Paraffin Embedded (FFPE) mouse tissue samples?
A: We do not test our antibodies in paraffin embedded tissue samples, because of the limited amount of published work with FFPE samples in the research community; however, researchers are welcome to try the antibodies in IHC of paraffin embedded tissues if they wish. Researchers are encouraged to publish their work on paraffin embedded tissues so that fellow researchers may be able to tell what antibodies do or do not work for this application.
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Q: Can I use eBioscience antibodies for immunoprecipitation (IP)?
A: If the products have been reported in the literature to work for IP, it will be so expressed in the product's Technical Data Sheet. eBioscience does quality control testing on selected products using immunohistochemical staining of frozen tissues, immunoprecipitation, and immunoblotting.
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Q: Can I use eBioscience antibodies for immunoblotting (WB)?
A: If the products have been reported in the literature to work for immunoblotting, it will be so expressed in the product's Technical Data Sheet. eBioscience does quality control testing on selected products using immunohistochemical staining of frozen tissues, immunoprecipitation, and immunoblotting.
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Q: I do not live in North America. Do you have any distributors in my country?
A: eBioscience has distributors worldwide, including Germany, Austria, Australia, Belgium, Croatia, China, Belgium, Denmark, Finland, France, Iceland, India, Italy, Japan, Korea, Luxemburg, Malayasia, Mexico, Netherlands, Norway, Singapore, Spain, Sweden, Switzerland, Taiwan, Thailand, Turkey, and United Kingdom.
Click here for contact information of our distributors. Customers can also order directly from our office in the United States.
Q: Why should I order from a distributor?
A: If you are in a country outside North America, packages sent to your country from the US may be subject to customs inspections. This delays shipping, subjecting many of our high quality products to longer periods in improper storage conditions. Ordering from a distributor may be more expensive, but avoids the shipping delays, which may potentially affect the performance of the products.
Q: If I order from eBioscience's US office, how long will it take for my order to arrive?
A: We normally ship the product via FedEx the day you place your order. If you are in North America, the shipment usually arrives at your shipping address a couple of days after we ship it. Packages sent to countries outside North America may take longer due to customs.
Q: Can you produce a custom product?
A: Yes, we perform custom product services regularly. We can perform custom conjugations and can produce our standard materials to meet your specific concentration or volume needs. To inquire, download our Custom Product Request Form and submit the completed form to customs@ebioscience.com. We will contact you within 2 business days of form submission regarding your inquiry.