Help & FAQs: Activity Assays

    • What is R&D Systems doing to reduce the use of packaging from non-renewable resources?
    • Environmental stewardship is important to R&D Systems. R&D Systems is ISO 14001:2015 Certified and as such, sustainability practices and "green" options are continuously reviewed. Recently, package inserts were removed from each Luminex kit in efforts to reduce waste. A QR code is now included on CoAs to direct end users to the appropriate insert. For other packaging materials, end users are encouraged to implement a recycling program locally. R&D Systems has chosen to reduce the use of styrofoam as much as possible with a variety of changes: 1. extensive stability testing in order to determine which products can be shipped with minimal packaging at ambient temperatures; 2. transitioning from the use of non-recyclable packaging materials to recyclable plastics, cardboard, or biodegradable materials; 3. continuing to investigate alternatives to dry ice shipments; and 4. the use of re-usable containers and gel packs that allow for smaller styrofoam containers.

    Annexin V Assays

    Apoptosis Detection Kits and Reagents

    • Are all of the components needed to run a TUNEL assay included in the kits that you offer?
    • The product datasheet for each kit lists all components supplied and any additional components required.
    • Does R&D Systems offer a protocol for apoptosis detection? 
    • The following protocol demonstrates an application of apoptosis detection with double labeling: https://www.rndsystems.com/resources/protocols/technical-notes-detection-apoptosis-double-labeling-tunel-and-active-caspase-3.  Additional protocols are available here: https://www.rndsystems.com/protocol-types/apoptosis-assays
    • How do I differentiate between apoptotic cells and necrotic cells when using Annexin V?
    • During early apoptosis phosphatidylserine is exposed on the cell surface. Annexin-V-FITC binds to phosphatidylserine and can be assayed by flow cytometry. Positive labeling is one to two logs higher than unlabeled cells. Propidium iodide can be added to the annexin-labeled cells to identify necrotic, late apoptotic, damaged, or dead cells exhibiting a compromised plasma membrane.
    • How does one discriminate between necrotic and apoptotic cells in TUNEL assays?
    • To discriminate apoptotic cells from necrotic cells, an additional method of apoptosis detection can be used. Immunoreactivity of active caspases within a cell, in addition to TUNEL staining, can indicate that a cell is apoptotic vs. necrotic. The following protocol demonstrates an application of apoptosis detection with this type of double labeling: https://www.rndsystems.com/resources/protocols/technical-notes-detection-apoptosis-double-labeling-tunel-and-active-caspase-3
    • What are apoptosis signalling pathways?
    • R&D Systems website has an interactive apoptosis signaling pathway webpage: https://www.rndsystems.com/pathways/apoptosis-signaling-pathway. 
    • What are TUNEL assays?
    • TUNEL assays are a method of labeling and detecting apoptotic cells. DNA fragments that are present in apoptotic cells can be detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). R&D Systems offers TUNEL-based kits designed for several different formats including Light Microscope, Fluorescence/Flow Cytometry, and Microplate Reader-based assays. We also offer colorimetric kits optimized for specific tissue types including Cardiac, Skin, Neuronal, Tumor, and Vascular cells and tissues.
    • What do TUNEL assays do?
    • TUNEL assays identify DNA fragmentation, a characteristic of both apoptotic as well as necrotic cells.
    • Why are my samples showing extensive cytoplasmic staining, in my Apoptosis Staining Kit? What does this mean?
    • Extensive cytoplasmic staining is indicative of high rate of cell death/late apoptosis or necrosis. To avoid this, reduce the time of your sample treatment in cell culture. This is because apoptosis in cell culture can progress to necrosis when given enough time.
    • Why does the TACS® Blue Label look green?
    • The green appearance is due to oxidation (presence of bleach, metals, and other oxidizing agents). Make sure to wash in deionized water before and after the TACS® Blue Label step. Ensure your work bench, pipettes, tips, etc are not contaminated with bleach or other strong oxidizing agents. Methyl Green may have been mistaken for TACS® Blue Label. Methyl Green can look blue prior to the ethanol washes and fades to green as excess dye washes away.
    • Why is Sybr Gold used in staining for CometAssay?
    • Important parameters to consider in choosing a DNA stain for the Comet assay are similar fluorescence and decay rates for single- and double-strand DNA. Sybr Gold has the desired features.
    • Why is TACS® Blue Label fading?
    • There are several possible reasons why TACS® Blue Label may fade after staining. Chlorine in tap water dissolves the blue label hence it is essential to use deionized water. Ensure proper dehydration through decreasing alcohol series (ethanol or denatured ethanol only), and o- or p-xylenes only (no mixed xylenes). Make sure to change solutions frequently. Use the correct mounting medium. The fading could also be due to benzene solubility. Benzene contaminants are found in some mixed xylenes. Use o- or p-xylenes for clarification after dehydration. Do not dilute mounting medium with mixed xylenes. Slides should be stored in the dark to maintain optimal staining. Please contact our technical service department for additional troubleshooting assistance.
    • Why is the TACS® blue stain being obscured by Nuclear Fast Red or Red Counter Stain C?
    • Nuclear Fast Red or Red Counter Stain C is taken up by some cells very rapidly. It is advisable to perform a background counterstain control to find the optimum incubation period with Nuclear Fast Red. (30 seconds - 5 minutes)
    • What is the difference between Cytonin™ and Proteinase K?
    • Customers should use either Proteinase K or Cytonin™ for cell permeabilization. Proteinase K is a robust permeabilization reagent and can compromise cell membrane integrity with long incubation periods. Cytonin™ is much gentler but may require optimization for some cell types and tissues.
    • Why are there two different types of TUNEL detection kits, TdT and TACS-XL, and which should I use?
    • There are two different TUNEL (terminal dUTP Nicked End Labeling) methods for apoptosis detection. The first uses TdT (Terminal deoxynucleotidyl Transferase) to incorporate biotinylated nucleotides into the 3'-OH ends of the DNA fragments. This labeling is optimized by the addition of a cation. The biotinylated nucleotides are then detected by using a streptavidin-HRP-conjugate followed by the substrate.The TACS-XL kits also use TdT to incorporate nucleotides into the 3'-OH ends of the DNA fragments. However, these nucleotides are BrdU-labeled rather than directly biotinylated. A biotinylated anti-BrdU antibody is then used for detection. More efficient incorporation of BrdUTP may result in improved signal-to-noise ratio.

    Caspase Activity Assays

    • Are R&D Systems Caspase Inhibitors irreversible?
    • Yes, the majority of R&D Systems Caspase Inhibitors have a Fluoromethyl ketone (FMK) functional group on the C-terminus of the peptide, and act as effective irreversible inhibitors with no added cytotoxic effects. Inhibitors synthesized with a benzyloxycarbonyl group (also known as BOC or Z) at the N-terminus and O-methyl side chains exhibit enhanced cellular permeability.R&D Systems also offers a General Caspase Inhibitor, Q-VD-OPh, Catalog # OPH001, as well as a FITC-conjugated pan-caspase inhibitor (ApoStat), Catalog # FMK012, which are both cell-permeable, irreversible inhibitors of caspase activity.
    • Are the Caspase Activity Assays specific for the only Caspase indicated on the insert?
    • The Caspase Activity Assays contain the substrate preferred by the individual Caspase being assayed. However, none of these substrates is completely specific for one Caspase. If more specificity is required, consider an ELISA which uses a specific capture antibody.
    • Does R&D Systems offer a positive control for the Caspase Activity Assays?
    • R&D Systems offers various recombinant Caspase enzymes that are ideal for use as positive controls. If one prefers to generate a positive control, there are several published methods. These include incubating cells with 2 µM Staurosporin for 2 hours, which induces apoptosis in most cell types. R&D Systems Technical Service has a list of references for generating positive controls for specific Caspases.
    • How much more sensitive is the Caspase-3 Fluorometric Assay Kit (Catalog # BF1100) than the Caspase-3 Colorimetric Assay Kit (Catalog # BF3100)?
    • No correlation regarding sensitivity has been tested comparing our fluorometric and colorimetric Caspase Activity Assays.
    • What is the molecular weight of AFC, the reporter molecule used in the Caspase Fluorometric Assays?  Can it be purchased separately?
    • The reporter molecule, 7-amino-4-trifluoromethyl coumarin (AFC), has a formula of C10H6NO2F3 with a molecular weight of 229.16. It is available as Catalog # B-210.
    • Why do I observe positive staining with Annexin-V FITC, but do not observe Caspase cleavage?
    • Annexin V-binding to phosphatidylserine in the outer leaflet is an early apoptotic event and cleavage of Caspase-3 occurs later. Therefore, a time-dependent study to determine the timecourse of different apoptotic events may be warranted.
    • Are the Caspase Activity Assays quantitative?
    • The Caspase Activity Assays are considered semi-quantitative. The results are best used to determine a fold increase in the Caspase activity in apoptotic cells over that of non-induced cells. The use of a background control (reactions where no cell lysate or substrate is added) is preferred. If this control gives a substantial reading, it should be subtracted from the experimental results prior to calculating the fold increase.
    • Can tissue homogenates be used with the Caspase kits (Catalog # BFxxxx)?
    • Yes, they can.  Homogenize the tissue using the cell lysis buffer, and incubate on ice for 10 minutes. Centrifuge to seperate and collect the cytosolic fraction for caspase activity determination.  If intending to store samples prior to assay, customers should determine if frozen storage is acceptable for their particular sample type.Customers are also recommended to consult the citations listed on the respective product pages for assistance in designing their tissue homogenate procedures with our Caspase kits.
    • How are the Caspase Activity Assays different from the Caspase ELISA Kits?
    • The Caspase ELISA Kits measure the total amount of Caspase present in the sample, regardless of the level of activity of the Caspase. These kits are more specific than the activity assays due to the use of antibodies for caspase recognition.The Capase Activity Assays offer a qualitative comparison of the Caspase activity in different samples, regardless of the total amount of Caspase. Specificity of Caspase enzymes are not absolute rather they are relatively specific.  Activity assays utilize the cleavage of a peptide as a measure of activity. Although a Caspase may have a preference for a particular peptide sequence, it may cleave similar sequences under the right conditions or given enough time.
    • How are the Caspase Activity Assays used?
    • Caspase Activity Assays provide a simple and convenient means for assaying protease activity in cell lysates. Upon cleavage of the Caspase-specific peptide substrate, a free reporter molecule can be quantified using a colorimetric or fluorometric microtiter plate reader. Comparison of the signal generated from an apoptotic sample with an uninduced control allows determination of the fold increase in Caspase activity. The level of Caspase activity is directly proportional to the signal detected.
    • Is the cell lysis buffer provided in the Caspase Activity Assay the same for all kits?
    • Yes, the cell lysis buffer supplied is the same for all Caspase Activity Kits. Therefore, cells can be lysed at one time, then used in multiple Caspase Activity Assays.

    CometAssay Single Cell Gel Electrophoresis Assays

    • Can cells isolated from urine be examined by CometAssay?
    • Sufficient cell count is necessary to run a successful CometAssay. The proper isolation of cells from urine would be necessary for optimal results.
    • Can the Alkaline CometAssay® Control Cells be used for the Neutral CometAssay?
    • The Alkaline CometAssay Control Cells were qualified for the Alkaline CometAssay while the Neutral CometAssay Control Cells were qualified for the Neutral CometAssay.
    • Can the CometChip® be stored before staining?
    • The neutralized gel can be stored in 20 mM Tris pH 7.4 buffer at 4 °C for a few days.
    • Do you recommend use of the CometAssay® with whole blood?
    • We do not recommend using whole blood with the CometAssay because sample preparation is critical, and hemoglobin could damage DNA. Additionally, it is important to note that vertebrate red blood cells (major blood component) do not have nuclei (except for birds) and therefore are not suitable for use with the CometAssay.
    • How do I avoid loss of agarose disks from the CometSlides™?
    • FLARE™ and CometSlides™ are specially treated to enhance agarose binding during alkaline treatment.Recommendations:Special attention needs to be paid to spread the agarose/cell mixture evenly over the entire slide well. Be careful to avoid getting agarose on the colored portion of the slides.Always place the slides into solutions; never pour or decant buffer over comet slides.Minimize time slides spend in alkaline buffer. Follow recommended incubation times.Rinse and suspend cells in PBS to remove medium before combining with LMAgarose.Do not increase ratio of cells to molten agarose by more than 1 to 10.Place slides at 4 °C for 15 min to assure complete gelling of the agarose before lysis.
    • How long can CometSlides™ be stored after electrophoresis?
    • Prior to staining, slides immersed in 70% ethanol for 5 minutes and then dried can be stored at room temperature for 1 year prior to staining. Permanent records can be created after staining for visualization by standard light microscopy using the CometAssay® Silver Staining Kit (Catalog # 4251-050-K).
    • How should the CometAssay® Control Cells be stored?
    • The CometAssay Control Cells should be stored in liquid nitrogen. To avoid the accumulation of damage due to freeze thaw cycles, the CometAssay Control Cells should be thawed and frozen in working aliquots. Refer to the CometAssay Control Cells literature for specific instructions.
    • Is it necessary to use a coverslip with CometSlides™?
    • FLARE™ and CometSlides™ were designed to be used without coverslips because removal of the coverslip could cause detachment of the agarose and cause damage to the samples.
    • Is it possible to combine the Anti-fade and SYBR® GOLD/SYBR® Green?
    • The solutions can only be mixed prior to application. Precipitation occurs upon storage of Anti-fade and SYBR mixture. 
    • Is it possible to re-use a CometSlide™ if not all wells are used?
    • CometSlides™ contain a special coating that promotes agarose binding, which can be compromised if washed for re-use.
    • Is it possible to reuse a CometChip® if not all wells are used?
    • The CometChip cannot be reused. After treatment and before lysis, the CometChip is covered with LMAgarose, which will fill the micropores in any unused wells.
    • Is it possible to treat cells in the CometChip® for longer than the recommended time?
    • The protocol will be reflective of what we have validated in-house for best results. If longer treatment times are required, the treatment step should be carried out in a tissue culture plate, after which time the cells can be transferred to the CometChip.
    • Is the Comet Assay Software (Catalog # 4260-000-CS) 21CFR certified?
    • No, the CometAssay software (Catalog # 4260-000-CS) is not 21CFR certified. We do not have a CometAssay software that is 21CFR certified.
    • Is there a convenient step to stop the CometAssay® and resume the next day?
    • The lysis protocol can be performed at 4 °C for 30-60 minutes or overnight. Otherwise, except for staining, the entire CometAssay® protocol must be completed until slides containing samples are completely dry. Slides may be stored at room temperature, with desiccant, prior to scoring at this stage.
    • The silver staining protocol for the CometAssay® requires 100 µL per well staining volume for two-well slides. What is the staining volume for twenty-well slides?
    • Silver staining volumes for the CometAssay can be reduced to 50 µL per well when using a twenty-well slide.
    • What are the dimensions of the CometSlides™?
    • The 2- and 3-well CometSlides have the same dimensions as standard microscope slides, 75 mm x 25 mm x 1 mm. The 20-well CometSlide™ is 75 mm x 50 mm x 1 mm. The 96-well CometSlide is 122 mm x 78 mm x 1 mm.
    • What can cause observation of artefacts during imaging of Comet data?
    • For optimal imaging, all cells should be in the same plane and this is achieved by completely drying the agarose. Artefacts may be introduced if the source cells used for analyis has any debris.  The debris should be allowed to settle before pipetting cells.  The LMagarose can also be the cause of artefacts.  To prevent debris from accumulating in the agar, melt it at 80-90 °C so it is runny, spin it at low speed and aliquot into sterile tubes in a hood. Use each aliquot for a single assay.
    • What cell line is used to generate the CometAssay® Control Cells?
    • CometAssay Control Cells were prepared using an immortalized human lymphocyte line.
    • What CometAssay® Electrophoresis Starter Kit is appropriate for use in Korea?
    • The CometAssay® Electrophoresis Starter Kit plus EU Power Supply (Catalog # 4250-050-ESK-PS2) is compatible with Korean outlets and voltage. Ensure voltage on the back of the power supply is set to 230 V before use.
    • What DNA damaging agent was used to create the CometAssay® Control Cells?
    • A healthy control cell population (C0) is treated with a DNA damaging agent to increase the amount of damage in populations C1, C2 and C3, respectively. Etoposide is the damaging agent used for Alkaline Control Cells (CC0-CC3) and Bleomycin is the damaging agent used for Neutral Control Cells (NC0-NC3).
    • What image analysis tools are available for analysis of data generated by the CometAssay®, and CometChip®?
    • We offer a software (Catalog # 4260-000-CS) designed to improve, standardize, and make comet analysis easier. The software will automatically locate and score comets from digital images to characterize and quantify the degree of DNA damage revealed by the comet assay. CometAssay, and CometChip Kits. The Comet Analysis Software can rapidly evaluate large numbers of cells and generate summary statistics based on the corresponding numeric results.
    • What is the difference between the CometChip® and the 96-well CometSlide™?
    • The 96-well CometSlide is employed in the traditional comet assay and requires each cell population to be treated and resuspended in molten LMAgarose individually before pipetting onto the CometSlide.In contrast, the CometChip uses gravity to capture cells in agarose pre-stamped with micropores for treatment directly within the slide, allowing for easier high throughput studies. After inserting the CometChip into the 96-well macroformer, cells are added to each well. Each macrowell of the CometChip contains ~400 non-overlapping micropores that capture a single cell for use in the comet assay. The cells can be treated, lysed, and electrophoresed directly on the CometChip.
    • What is the importance of pH for the alkaline electrophoresis buffer in the Comet Assay?
    • The pH of the electrophoresis buffer will determine the types of DNA damage that can be analyzed. At pH 12.1, initial breaks are analyzed, while at pH 12.5 and pH 13 alkaline labile adducts are converted to breaks. At pH 12.5, basic lesions are converted to single strand breaks and at pH 13, additional labile sites are converted to single and double strand breaks. Maximum damage caused by an agent is visualized at pH 13 in FLARE™ Assay, CometAssay®, and CometChip® Kits.
    • What is the optimal filter set for SYBR® GOLD?
    • SYBR® Gold’s maximum excitation/emission is 496 nm/522 nm. A fluorescein filter should be adequate.
    • What is the purpose of the CometAssay® Control Cells?
    • The CometAssay Control Cells are designed to help standardize and compare alkaline and neutral electrophoresis methods between individual users and laboratories. The control cells contain known levels of damage.
    • What is the size of a single pore in the CometChip®?
    • The CometChip micropores are 30 µm in diameter and 50 µm deep.
    • What staining alternatives to SYBR® Gold are suitable for the CometAssay®?
    • SYBR Gold nucleic acid gel stain (10,000X concentrate in DMSO) is recommended. SYBR Green can also be used.Less sensitive alternatives include the following:Acridine Orange (50 µM)- single strand breaks appear red and double strand breaks appear yellow–green.Ethidium Bromide (20 µg/ml) –binds double strand breaks more efficiently than single strand breaks.DAPI (1 µg/ml) - binds to major groove of DNA and fluorescence is dependent on double-stranded structure.Propidium Iodine (2.5 to 20 µg/ml) – binds to DNA by intercalating between bases.Hoechst 33258 (0.5 µg/ml) – binds to minor groove of DNA.YoYo-1 – binds to DNA by intercalating between bases.  R&D Systems has not tested YoYo-1 in house, so there is no recommended concentration.
    • Why are comet tails in positive control cells not present or smaller than expected?
    • Failure to lyse, denature in alkali, or properly perform electrophoresis may generate poor results.  To improve results:Verify lysis of cells. Lysis Solution should be stored long term at room temperature and chilled to 4 °C prior to use. Lysis Solution stored long term at 4 °C will precipitate. Try overnight lysis at 4 °C.Perform alkali denaturation for 20 minutes at room temperature or for at least 1 hour at 4 °C. Prepare Alkaline Solution fresh from sodium hydroxide pellets.Electrophoresis conditions are also critical for optimal performance. Conventional slab gel electrophoresis chambers are not designed to eliminate known causes of comet assay variability (alkaline pH, buffer height, temperature, slide orientation). For optimal results, use a CometAssay Electrophoresis system (Catalog # 4250-050-ES). For insufficient electrophoresis time, increase time up to 1 hour at 4 °C for alkaline electrophoresis.
    • Why are there comet tails in the healthy CometAssay® Control Cell population?
    • The CometAssay Control Cells were prepared from an asynchronous cell population with cells at all stages of the cell cycle. Some percentage of the population will be undergoing DNA replication and may have strand breaks at replication forks.Improper storage may also lead to DNA damage. Do not store CometAssay Control Cells at -80°C. Store in aliquots in liquid nitrogen.
    • Why don't I see a visible cell pellet after centrifugation of the CometAssay® Control Cells?
    • A pellet may not be visible after centrifugation due to the low number of cells.
    • Why is the cell culture medium leaking out from the wells of the CometChip® macroformer?
    • There are a few things that may lead to leaking in the macroformer:Treatment time is too long. The macroformer is designed for ~30 minute loading time followed by ~1 hour treatment time. Longer treatment times may result in leaking. If longer treatments are required, the treatment step should be carried out in a tissue culture plate, after which time the cells can be transferred to the CometChip.The slide was not properly placed into the macroformer. If the slide is not positioned properly on the black bottom, the macroformer will not snap into place to properly make a seal. Ensure that the macroformer is evenly spaced above the black base. Always carry the unit by the black base. If held by the white macroformer, the magnets will separate and cause leakage.The CometChip is expired. The CometChip has an 8-week shelf life. Expired CometChips begin to dry out and will not form a proper seal with the macroformer.
    • Why is it necessary to rinse the CometAssay® Control Cells in PBS before adding to LMAgarose?
    • The CometAssay Control Cells must be rinsed in PBS to eliminate media and storage factors that reduce adherence of LMAgarose to the slide. PBS without magnesium should be used, as magnesium stimulates nucleases.
    • Will a ZEISS Axioplan/Axiovert microscope work for viewing CometSlides™?
    • Yes, the ZEISS Axioplan/Axiovert microscope will work for viewing a FLARE™ or CometSlide™.
    • Will any commercial horizontal gel electrophoresis device work for the Comet Assay?
    • Electrophoresis conditions described for FLARE™ Assay, CometAssay® and CometChip® Kits were optimized for the CometAssay® Electrophoresis System (Catalog # 4250-050-ES). Conventional slab gel electrophoresis chambers are not designed to eliminate known causes of comet assay variability (alkaline pH, buffer height, temperature, slide orientation). They can be used, but they require optimization by the user to achieve consistent results.

    FLARE Assay Kits

    • What is the importance of pH for the alkaline electrophoresis buffer in the Comet Assay?
    • The pH of the electrophoresis buffer will determine the types of DNA damage that can be analyzed. At pH 12.1, initial breaks are analyzed, while at pH 12.5 and pH 13 alkaline labile adducts are converted to breaks. At pH 12.5, basic lesions are converted to single strand breaks and at pH 13, additional labile sites are converted to single and double strand breaks. Maximum damage caused by an agent is visualized at pH 13 in FLARE™ Assay, CometAssay®, and CometChip® Kits.