FAQs

CategoryQuestionAnswerCatalog Number
Oxidative StressWhat assays can be performed in order to obtain a more quantitative assessment of how much oxidative stress is present in my experimental system?8-OHdG is a frequently used biomarker of oxidative DNA damage and oxidative stress. For detection by immunocytochemistry or immunohistochemistry, we recommend using our 8-oxo-dG 2E2 monoclonal antibody. Please see protocol

Trevigen also offers a validated HT 8-oxo-dG ELISA kit II, a fast and sensitive immunoassay for the detection and quantitation of 8-OHdG in DNA, plasma, urine, and saliva samples. Results can be expressed in quantitative terms. Please see:
4380-096-K
Oxidative StressWhat assay do you offer for detection of superoxide radicals?For colorimetric detection of superoxide radical, we offer our Superoxide Dismutase Assays. We have an HT 96 Well Plate version (7501-500-K) and a cuvette version (7500-100-K) to fit your needs.7500-100-K, 7501-500-K
Oxidative StressDoes the HT Glutathione Assay kit (7511-100-K) distinguish between oxidized and reduced glutathione?Yes, the HT Glutathione Assay kit does measure total glutathione as well as oxidized and reduced glutathione.

The addition of 4-vinylpyridine to the standard or cell extract blocks reduced glutathione (GSH) but not oxidized glutathione (GSSG). Thus the only form remaining after 4-vinylpyridine treatment is GSSG. The reduced form (GSH) is calculated by first determining total glutathione with the kit, and then treating with 4-vinylpyridine. Subtracting GSSG from total glutathione then gives the reduced GSH concentration.
7511-100-K
Oxidative StressAre the SOD kits sensitive enough to detect SOD in Human serum or plasma samples?The SOD Assay kits are suitable for mammalian cell extracts. The SOD concentration in plasma or serum is very low, and may not be detectable.7500-100-K, 7501-500-K
Oxidative StressHow do you differentiate between the different forms of SOD?The SOD assay can differentiate between the various SOD isozymes: SOD1 (cytosolic Cu/Zn-SOD), SOD2 (mitochondrial Mn-SOD), and SOD3 (extracellular Cu/Zn-SOD).

1. SOD2 can be inactivated by adding 400 µL or 800 µL; of ice-cold chloroform/ethanol (37.5/62.5 (v/v)) to 250 µL of erythrocyte lysate or 500 µL of cell/tissue lysate, respectively, shaking for 30 sec, and then centrifuging at 2,500 x g for 10 min. Assay the aqueous upper phase for SOD1 immediately or freeze in aliquots at -80°C.

2. The addition of cyanide ion to a final concentration of 2 mM inhibits more than 90% of SOD1 and SOD3 activity. SOD2 is unaffected by cyanide.

3. SOD3 is isolated from the extracellular matrix of tissue. SOD3 has been found in serum and in cerebrospinal, ascetic, and synovial fluids.
7500-100-K, 7501-500-K
PARP, PARG and TankyraseWhy shouldn't I use the PARP Universal Assay Kits to measure PARP inhibition in cells or tissue samples?To measure PARP1 activity within cultured cells, tissue and tumor lysates, we recommend our sensitive PARP in vivo Pharmacodynamic Assay Kit. The Universal Assay Kits are designed for the in vitro verification of lead compounds with purified PARP1 and determination of IC50 values. The dilution of lysates and subsequent assay of PARP activity in the Universal Assay Kits, allows for dissociation and dilution of test PARP inhibitors, leading to absence of inhibition in the assay, even though inhibition may have been present in the cells, prior to lysis.

For PARP kit selection guidance with recommended use information, please see our selection graphic. https://trevigen.com/product-category/cell-stress-and-dna-damage/dna-damage/cell-stress-and-dna-damage-dna-damage-parp-parg-and-tankyrase/cell-stress-and-dna-damage-dna-damage-parp-parg-and-tankyrase-kits/.
4520-096-K, 4676-096-K, 4677-096-K
PARP, PARG and TankyraseDoes PARP1 recognize a specific type of DNA damage?PARP1 predominantly recognizes single strand breaks. 4668-100-01, 4668-500-01,
4668-02K-01
PARP, PARG and TankyraseWhat is the final NAD concentration in the PARP Universal Assay Kits?The final NAD concentration (including biotinylated NAD) in the PARP Universal Assay Kits is 68 µM.4676-096-K,
4677-096-K
PARP, PARG and TankyraseIs there a substitute stop solution for the colorimetric reaction instead of 0.2M HCl in the PARP Universal Colorimetric Assay Kit?5% phosphoric acid can be used in place of 0.2M HCl to stop the colorimetric reaction.4677-096-K
PARP, PARG and TankyraseDo the PARP/Apoptosis Assay Kits measure all possible PARP activity in cell extracts?The PARP/Apoptosis Assay Kits will measure the net activity of PARP1 in cell extracts. However, if testing for in vivo inhibition of PARP activity use the PARP in vivo Pharmacodynamic Assay Kit.4520-096-K,
4684-096-K,
4685-096-K
PARP, PARG and TankyraseWhat is the absorbance reading (blue versus yellow color) for analysis in the PARP Universal Colorimetric Assay Kit? TACS-Sapphire® is a Horseradish Peroxidase (HRP) substrate generating a soluble blue color with maximum absorbance at 630 nm (range 620-650). Development of the colorimetric reaction can be stopped by the addition of an equal volume of 0.2 M HCl or 5% phosphoric acid, generating a yellow color which is stable for up to 60 minutes and can be read at 450 nm (range 440-460). The absorbance at 450 nm will be significantly higher than the absorbance at 630 nm.4677-096-K
PARP, PARG and TankyraseWill the Anti-PAR Monoclonal and Polyclonal Antibodies recognize mono-ADP ribosylated proteins?No, the Anti-PAR Monoclonal Antibody (cat# 4335-MC-100) will not recognize ADP-ribose monomers. This antibody was raised against purified PAR polymer chains of 10 to 50 units long; therefore, it is only specific for poly (ADP-ribose) polymers.

The Anti-PAR Polyclonal Antibody (cat# 4336-BPC-100) was raised against poly (ADP-ribose) polymer and recognizes free poly (ADP-ribose) and poly-ribosylated proteins. The antibody has not been tested for recognition of mono-ADP ribosylated proteins.
4335-MC-100, 4336-BPC-100
PARP, PARG and TankyraseWhat is the difference between Anti-PAR and Anti-PARP antibodies?Anti-PAR antibody recognizes poly ADP-ribose (PAR polymer) while the Anti-PARP antibody recognizes the PARP1 enzyme that synthesizes the PAR polymer.4335-MC-100, 4336-BPC-100, 4338-MC-50
Comet AssayWill comet assay work with solid tissue? If so how?Prepare single-cell suspensions from tissues as described below if using FLARE™ Assay or CometAssay® Kits. Using 50 µl of a cell suspension at 1 x 105 cells per ml combined with 500 µl of LMAgarose will provide the correct agarose concentration and cell density for optimal results when spreading 50 µl per well using CometSlide™ 2 Well.

Tissue Preparation
Place a small piece of tissue into 1–2 ml of ice cold 1X PBS (Ca++ and Mg++ free), 20 mM EDTA. Using small dissecting scissors mince the tissue into very small pieces and let stand for 5 minutes. Recover the cell suspension, avoiding transfer of debris. Count cells, pellet by centrifugation, and suspend at 1 x 105 cells/ml in ice cold 1X PBS (Ca++ and Mg++ free).

For blood rich organs (e.g., liver, spleen), chop tissue into larger pieces (1–2 mm3), let settle for 5 minutes then aspirate and discard medium. Add 1–2 ml of ice cold 20 mM EDTA in 1X PBS (Ca++ and Mg++ free), mince the tissue into very small pieces and let stand for 5 minutes. Recover the cell suspension, avoiding transfer of debris. Count cells, pellet, and suspend at 1 x 105 cells/ml in ice cold 1X PBS (Ca++ and Mg++ free).
4040-100-FK,
4045-01K-FK,
4130-100-FK,
4250-050-K,
4251-050-K,
4252-040-K,
4253-096-K
Comet AssayWhat is the optimal filter set for SYBR® GOLD?SYBR ® Gold’s maximum excitation/emission is 496 nm/522 nm. Fluorescein filter is adequate.4040-100-FK,
4045-01K-FK,
4130-100-FK,
4250-050-K,
4252-040-K,
4253-096-K
4260-096-CSK
Comet AssayWhat image analysis tools are available for scoring comets?Trevigen now offers a breakthrough software (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. It is compatible with FLARE™ Assay, CometAssay®, and CometChip® Kits. Trevigen’s Comet Analysis Software can rapidly evaluate large numbers of cells, related to each treatment group or screening target in a study, and generate summary statistics based on the corresponding numeric results.4040-100-FK,
4045-01K-FK,
4130-100-FK,
4250-050-K,
4252-040-K,
4253-096-K,
4260-096-CSK, 4260-000-CS
Comet AssayWhy is there no evidence of comet tail in the positive control (ex. hydrogen peroxide)?Failure to lyse, denature in alkali, or to properly perform electrophoresis may generate poor results.

Recommendations for CometAssay® Kits:
1) Try overnight lysis at 4°C.
2) Perform alkali denaturation for 20 minutes at room temperature or for at least 1 hour at 4°C.
3) Prepare Alkaline Solution fresh from sodium hydroxide pellets.
4) Use fresh hydrogen peroxide to induce damage.
4250-050-K,
4251-050-K,
4252-040-K,
4253-096-K
Comet AssayWhat is the importance of pH for the alkaline electrophoresis buffer?The answer will depend upon DNA damage adducts under analysis. 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, abasic 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.
4040-100-FK,
4045-01K-FK,
4130-100-FK,
4250-050-K,
4251-050-K,
4252-040-K,
4253-096-K,
4260-096-CSK
Comet AssayWhy are negative controls showing more DNA damage than expected in adherent cells?1)      Reduce cell exposure to Trypsin or try alternative detachment methods.

Suggestions:
10-minute incubation at 37°C in PBS/1 mM EDTA followed by 2-minute Trypsin treatment.
30 minute incubation at 4°C in cold Trypsin.
Cold EDTA (2 mM) can be used instead of Trypsin to detach cells.
Detach cells by scraping using a rubber policeman.
Centrifuge cells for 10 minutes at speeds less than 200xg to avoid DNA damage.
4040-100-FK,
4045-01K-FK,
4130-100-FK,
4250-050-K,
4251-050-K,
4252-040-K,
4253-096-K,
4260-096-CSK
Comet AssayWhat is the effect of light on the comet assay?The samples can be handled under normal laboratory lights but the Lysis and Alkali Unwinding Steps should be performed in the dark. Electrophoresis is typically performed using a system such a Trevigen’s CometAssay® Electrophoresis System (4250-050-ES) designed to minimize exposure to UV light.
4040-100-FK,
4045-01K-FK,
4130-100-FK,
4250-050-K,
4251-050-K,
4252-040-K,
4253-096-K,
4260-096-CSK
Comet AssayHow long can FLARE™ Slides or CometSlides™ be stored?Prior to staining with SYBR™ Gold, dried slides stored with desiccant can be kept for extended periods (months).

Using the CometAssay® Silver Staining Kit (4251-050-K), permanent records are created and visualization using standard light microscopy is possible.
4040-100-FK,
4045-01K-FK,
4130-100-FK,
4250-050-K,
4251-050-K,
4252-040-K,
4253-096-K
Comet AssayDoes it matter if the same pH is used for alkali unwinding and alkaline electrophoresis?The same solution (> pH 13) is used for alkali unwinding and alkaline electrophoresis. To avoid variation due to pH, the buffers should be prepared fresh.

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, abasic 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 CometAssay® and FLARE™ Assay.
4040-100-FK,
4045-01K-FK,
4130-100-FK,
4250-050-K,
4251-050-K,
4252-040-K,
4253-096-K
Comet AssayIs it necessary to use a coverslip with comet assay slides?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.

4040-100-FK,
4045-01K-FK,
4130-100-FK,
4250-050-K,
4251-050-K,
4252-040-K,
4253-096-K
Comet AssayIs it possible to combine the Anti-fade and SYBR® GOLD?The solutions can only be mixed prior to application. Precipitation occurs upon storage of Anti-fade and SYBR® Gold mixture.4040-100-FK,
4045-01K-FK,
4130-100-FK,
4250-050-K,
4252-040-K,
4253-096-K
Comet AssayDo you recommend use of the comet assay with whole blood?We do not recommend using whole blood with the comet assay because sample preparation is critical and hemoglobin could damage DNA. It's important to note that vertebrate red blood cells (major blood component) do not have a nucleus (except for birds) and therefore not suitable for use with the CometAssay®.4040-100-FK,
4045-01K-FK,
4130-100-FK,
4250-050-K,
4251-050-K,
4252-040-K,
4253-096-K,
4260-096-CSK
Comet AssayHow long can FLARE™ Slides or CometSlides™ be stored before applying SYBR®GOLD Staining Solution?Slides immersed in 70% ethanol for 5 min and dried can be stored at room temperature for 1 year prior to staining.4040-100-FK,
4045-01K-FK,
4130-100-FK,
4250-050-K,
4252-040-K,
4253-096-K
Comet AssayIs there a convenient step to stop the comet assay and resume the next day due to time constraints?The lysis protocol can be performed at 4 ° C for 30-60 minutes or overnight. Otherwise, except for staining, the entire comet assay protocol must be completed until slides containing samples are completely dry. Slides may then be stored at room temperature, with desiccant, prior to scoring at this stage.4040-100-FK,
4045-01K-FK,
4130-100-FK,
4250-050-K,
4251-050-K,
4252-040-K,
4253-096-K
Comet AssayWhy are comet tails in positive control cells smaller than expected?Electrophoresis times for each slide type are provided for the CometAssay® Electrophoresis System (4250-050-ES). For other systems, the electrophoresis time must be optimized.

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.
4040-100-FK,
4045-01K-FK,
4130-100-FK,
4250-050-K,
4251-050-K,
4252-040-K,
4253-096-K,
4260-096-CSK
Comet AssayIs it possible to re-use a comet assay slide if not all wells are used ?FLARE™ and CometSlides™ contain a special coating for binding the agarose, which can be compromised if washed for re-use.4040-100-FK,
4045-01K-FK,
4130-100-FK,
4250-050-K,
4251-050-K,
4252-040-K,
4253-096-K
Comet AssayThe silver staining protocol for the comet assay 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 comet assay can be reduced to 50 µl per well when using a 20-well slide.4251-050-K,
4254-200-K
Comet AssayWill a ZEISS Axioplan/Axiovert microscope work for viewing comet slidesThe ZEISS Axioplan/Axiovert microscope will work for viewing a FLARE™ or CometSlide™.4040-100-FK,
4045-01K-FK,
4130-100-FK,
4250-050-K,
4251-050-K,
4252-040-K,
4253-096-K
Comet AssayIs it possible to differentiate between apoptotic and necrotic cells using the comet assay?Listed below are two references discussing differentiation of apoptotic and necrotic cells using a derivation of the comet assay.

Fairbairn, D. W., Walburger D. K, Fairbairn J. J and O'Neill K. L. Key morphologic changes and DNA strand breaks in human lymphoid cells: discriminating apoptosis from necrosis. Scanning 1996 Sep: 18(6):407-16

Abstract

Apoptosis is an important form of physiologic cell death displayed by an enormous variety of tissues under divergent conditions. The recent attention toward apoptosis in virtually all aspects of modern biology indicates that rapid and accurate differentiation between apoptosis and necrotic death is of considerable interest. Apoptosis is distinguishable from necrosis on the basis of several criteria. In this study, we undertook to examine the effects of mild hyperthermia (43 degrees C leading to apoptotic death) and severe hyperthermia (50 degrees C leading to necrotic killing) on associated DNA fragmentation. Using laser scanning and fluorescent microscopic evaluation of DNA “comets” in the single cell gel assay, we compared necrotic and apoptotic DNA damage in a variety of human leukemia and lymphoma cell lines at the level of the individual cell. We show that necrotic cells do display detectable DNA damage. We confirm our preliminary report that comet “tail moment” is sufficient to distinguish between necrotic and apoptotic DNA damage, while comet tail length may confuse the two forms. We report that recovery period is necessary for expression of increasing apoptotic but not necrotic DNA damage. We show that apoptosis increases with prolonged hyperthermia and confirm that the mode of death changes from apoptosis to necrosis with higher heat loads, producing a greater fraction of cells showing damage. In addition, we show that for necrotic cells, DNA tail moment reflects sensitivity to prolonged exposure without a concomitant change in tail length.

Singh, N.P., A simple method for accurate estimation of apoptotic cells. Exp Cell Res 2000 Apr 10; 256(1):328-37

Abstract

A simple sensitive, and reliable “DNA diffusion” assay for the quantification of apoptosis is described. Human lymphocytes and human lymphoblastoid cells, MOLT-4, were exposed to 0, 12.5, 25, 50, or 100 rad of X-rays. After 24h of incubation, cells were mixed with agarose, microgels were made, and cells were lysed in high salt and detergents. DNA was precipitated in microgels by ethanol. Staining of DNA was done with an intense fluorescent dye, YOYO-1. Apoptotic cells show a halo of granular DNA with a hazy outer boundary. Necrotic cells, resulting from hyperthermia treatment, on the other hand, show an unusually large homogeneous nucleus with a clearly defined boundary. The number of cells with apoptotic and necrotic appearance can be scored and quantified by using a fluorescent microscope. Results were compared with other methods of apoptosis measurement: morphological estimations of apoptosis and DNA ladder pattern formation in regular agarose gel electrophoresis. Validation of the technique was done using some known inducers of apoptosis and necrosis.
4250-050-K,
4252-040-K,
4253-096-K
Comet AssayWhat staining alternatives to SYBR® Green I are suitable for the comet assay?SYBR® Gold nucleic acid gel stain (10,000X concentrate in DMSO) is recommended.

Less sensitive alternatives
1. Acridine Orange (50 µM)- single strand breaks appear red and double strand breaks appear yellow–green.
2. Ethidium Bromide (20 µg/ml) –binds double strand breaks more efficiently than single strand breaks.
3. DAPI (1 µg/ml) - binds to major groove of DNA and fluorescence is dependent on double-stranded structure.
4. Propidium Iodine (2.5 to 20 µg/ml) – binds to DNA by intercalating between bases
5. Hoechst 33258 (0.5 µg/ml) – binds to minor groove of DNA
4040-100-FK,
4045-01K-FK,
4130-100-FK,
4250-050-K,
4252-040-K,
4253-096-K
Comet AssayHow do I avoid loss of agarose disks from the comet slides?1.       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 agarose on the colored.
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 agar by more than 1 to 10.
Place slides at 4°C for 15 min to assure complete gelling of the agar before lysis.
4040-100-FK,
4045-01K-FK,
4130-100-FK,
4250-050-K,
4251-050-K,
4252-040-K,
4253-096-K,
4256-010-CC,  
4257-010-NC
Comet AssayWill 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 (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. 4040-100-FK,
4045-01K-FK,
4130-100-FK,
4250-050-K,
4250-050-ES
4251-050-K,
4252-040-K,
4253-096-K,
4260-096-CSK
Comet AssayHow 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, the CometAssay® Control Cells should be thawed and frozen in working aliquots.4256-010-CC,  
4257-010-NC
Comet AssayWhat cell line is used to generate the CometAssay® Control Cells?CometAssay® Controls were prepared using an immortalized human lymphocyte line.4256-010-CC,  
4257-010-NC
Comet AssayWhat 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).4256-010-CC,  
4257-010-NC
Comet AssayCan the Alkaline CometAssay® Control Cells be used for the neutral comet assay?The Alkaline CometAssay® Control Cells were qualified for the alkaline comet assay while the Neutral CometAssay® Control Cells were qualified for the neutral comet assay.4256-010-CC,  
4257-010-NC
Comet AssayHow are the CometAssay® Control Cells to be used?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.4256-010-CC,  
4257-010-NC
Comet AssayIs it necessary to rinse the CometAssay® Control Cells in PBS before adding to LMAgarose?Yes, the CometAssay® Control Cells must be rinsed in PBS to eliminate media and storage factors that reduce adherence of LMAgarose to the slide.4256-010-CC,  
4257-010-NC
Comet AssayWhy don't I see a visible cell pellet after centrifugation of the CometAssay® Control Cells?A pellet will not be visible after centrifugation due to the low number of cells.4256-010-CC,  
4257-010-NC
Comet AssayWhy don't I see comets after staining the CometAssay® Control Cells?After cell centrifugation, leave ~50 µl of supernatant to avoid the loss of cells while pipetting off the supernatant. If necessary, centrifugation speeds can be increased to 500xg.4256-010-CC,  
4257-010-NC
Comet AssayWhy 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.

Do not store CometAssay® Control Cells at -80°C. Store in aliquots in Liquid Nitrogen.
4256-010-CC,  
4257-010-NC
Comet AssayWhat would cause my treated CometAssay® Control Cells not to show Comet tails?Failure to lyse, denature in alkali, or to properly perform electrophoresis may generate poor results.

Prepare fresh Alkaline Solution from NaOH pellets and increase time in alkaline solution up to 1 hour at 4°C.

Electrophoresis conditions described for CometAssay® Control Cells were optimized for the CometAssay® Electrophoresis System (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.
4250-050-ES,
4256-010-CC,  
4257-010-NC
Comet AssayWhat would cause Comet tails to be present, but not significant in treated control cells?Electrophoresis conditions described for CometAssay® Control Cells were optimized for the CometAssay® Electrophoresis System (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. For insufficient electrophoresis time, increase time up to 1 hour at 4®C for alkaline electrophoresis.
4250-050-ES,
4256-010-CC,  
4257-010-NC
Annexin V kits and related productsWhat is Propidium Iodide (PI) Propidium Iodide is a fluorescent DNA intercalating dye used as an indicator of plasma membrane integrity. In healthy cells, PI cannot cross the plasma membrane, but once the membrane becomes compromised, PI enters the cell and intercalates into the DNA. When used in conjunction with labeled Annexin V, PI allows the distinction between early and late apoptotic/necrotic cells populations.4830-01-K
4830-250-K
4835-01-K
4835-250-K
Annexin V kits and related productsWhat is Annexin V-FITC Annexin V is a calcium-dependent phospholipid binding protein that can be used to bind phosphatidylserine (PS). In normal cells, phosphatidylserine is sequestered along the inner surface of the cytoplasmic membrane. During apoptosis, PS redistributes along the outer surface of the membrane due to caspase inactivation of the PS flippase enzyme. Binding of FITC-labeled Annexin V to PS provides a fluorescent detection option for PS externalization and is an indicator of apoptosis.4830-01-K
4830-250-K
Annexin V kits and related productsCan Annexin V-FITC be used with cells expressing green fluorescent protein (GFP)?TACS® Annexin V-FITC kit cannot be used with cells expressing GFP since the fluorescence molecules have similar excitation and emission spectra. However, the TACS® Annexin V-Biotin Kit offers flexibility allowing use of other fluorophores. 4830-01-K
4830-250-K
4835-01-K
4835-250-K
Cell proliferation kitsIs the MTT Detergent Reagent toxic to my cells?The Detergent Reagent has sufficient SDS to disrupt the cell membrane and is therefore toxic to cells. The role of the Detergent Reagent is to solubilize the formazan dye prior to measuring the absorbance.4890-25-K
4890-50-K
TUNEL based kitsProcedure of HT TiterTACS Assay kit (Cat # 4822-96-K). Are the centrifuge between steps required for the cells in monolayer in 96 well plate?IF yes why?'Including the centrifugation steps in the TiterTACS Assay kit (Cat # 4822-96-K) is highly recommended as well for monolayer cells to avoid sample loss that can occur during washes.
4822-96-K
TUNEL based kitsWhat is the right cation for my sample?Please consult the reference table in appendix C of the instruction manual for 4811-30-K.
https://trevigen.com/docs/protocol/protocol_4810-30-K.pdf
These recommendations were compiled from past labeling experiments as well as collaborators publications.
All specialty TACS kits
TUNEL based kitsDo the TACS 2 TdT Kits work for Rabbit samples?The TACS 2 TdT Kits do not have any limitations due to sample species.All specialty TACS kits
TUNEL based kitsWhat is the difference between Cytonin® and Proteinase K?Customers should choose to use either Proteinase K or Cytonin® to permeabilize their samples. Proteinase K is a robust, enzymatic permeabilization reagent that is frequently used but could compromise cell membrane integrity with long incubation periods. Cytonin® is much gentler but may require optimization for some cell types and tissues. All specialty TACS kits
TACS- XL
TUNEL based kitsWhich TACS® Kit is recommended for retinal tissues?To detect apoptotic cells in retina by TUNEL method, the TACS® 2 TdT DAB Kit is recommended. In-house labeling of rat retina (fresh frozen tissue) using the TACS®2 TdT DAB gives excellent results when the samples are treated with Cytonin or a 1:200 dilution of Proteinase K for 10 minutes, and compared to a positive control treated with TACS-Nuclease.4810-30-K
TUNEL based kitsWhich method or kit is recommended to detect apoptosis in frozen brain (hippocampus) samples from a heat stressed animal? NeuroTACS™ II In Situ Apoptosis Detection Kit is recommended for convenient identification of apoptosis in brain tissue or neuronal cells.4823-30-K
TUNEL based kitsIs it recommended that Trevigen’s Mounting Media be used with TACS® In Situ Apoptosis Detection kits using either diaminobenzidine or TACS Blue Label®? It is important to use Mounting Media free of ortho-, para- and mixed xylenes. Xylenes result in fading of TACS Blue Label®.

It is possible that fading of Blue Label may occur with some batches of Permount due to presence of xylenes. DPX is advertised as free of xylenes. An alternate is butyl-acetate acrylic mixture from Polysciences, which is fast drying and is more likely to hold the color of the Blue Label.
All specialty TACS kits
TACS- XL
TUNEL based kitsWhy is TACS Blue Label® fading?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-xylene only (no mixed xylenes). Make sure to change solutions frequently.

Use correct mounting medium. Trevigen Mounting Media (cat# 4865-25) is the recommended mounting medium. The fading could also be due to benzene solubility. Benzene contaminants are found in some mixed xylenes. Use o- or p- xylene for clarification after dehydration. Do not dilute mounting medium with mixed xylenes.

Slides should be stored in the dark to maintain optimal staining.
4811-30-K
4828-30-BK
4827-30-K
4829-30-K
TUNEL based kitsWhy does the blue label appear very weak?Insufficient wash steps. Make sure to wash in deionized water before and after the TACS Blue Label® step.

Labeling reaction time was insufficient. View under microscope to determine proper incubation period with TACS Blue Label? (2-7 minutes).
4811-30-K
4828-30-BK
4827-30-K
4829-30-K
TUNEL based kitsUsing TACS 2 TdT Blue Label kit, why are there large clumps of blue precipitates on my slide?The length of the colorimetric reaction with TACS Blue Label was too long. Reducing the incubation time should provide a localized staining.4811-30-K
4828-30-BK
4827-30-K
4829-30-K
TUNEL based kitsWhy is my 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)4811-30-K
4828-30-BK
4827-30-K
4829-30-K
TUNEL based kitsWhy does the DAB solution appear milkyThe DAB solution tends to precipitate when cold. Prewarm the solution as directed in the protocol4811-30-K
4828-30-DK
TUNEL based kitsDo you have a procedure for in situ labeling of apoptotic cells in bone and cartilage?Labeling of apoptotic cells with the TACS 2 TdT DAB kit was published in Experimental & Molecular Medicine (2016) 48, e256; doi:10.1038/emm.2016.75.
http://www.nature.com/emm/journal/v48/n9/full/emm201675a.html

Decalcification is required for bone and cartilage. Treatment of tissue will vary with size, porosity, and type of tissue. It is important to note that bone and cartilage can give high background in these assays due to poor quality or tissues. For example, damage to samples can occur with incomplete fixation, roughened blade during sectioning, or strong proteinase K treatment.

Protocol Guideline:
1. Tissues should be fixed in 4% paraformaldehyde (10% neutral buffered formalin or 2-4% formaldehyde prepared in PBS) for 8 to 12 hours with one or two changes of paraformaldehyde solution. It may be necessary to gently separate the joint with dissection tools, while in fixative, to facilitate fixation throughout the entire tissue.

2. After fixation, the tissue is placed into 10 volumes of Decalcification Solution (recipe below) for up to 3 weeks at room temperature. It may be necessary to change the Decalcification Solution several times during this period. Decalcification should be allowed to continue until the tissue is pliable for sectioning. If further decalcification is required, it is possible to further decalcify sectioned tissues on slides.

3. Tissue should be placed into increasing ethanol series (70%, 95%, 100%) and then xylene prior to embedding in paraffin.

4. Sections should be cut at 5 µm, and floated onto glass slides treated for electrostatic adherence. Sections should be dried at 45°C for up to one hour or at room temperature overnight. Slides are ready for labeling at this point.

Decalcification Solution:
To prepare two liters:
250g EDTA, disodium salt
25g sodium hydroxide
Make solution to 2 liters (~1750 mL deionized water)
pH should be approximately 7.0
4811-30-K
4828-
TUNEL based kitsDo you have a supplementary protocol for LR White Embedded Tissue (Electron Microscopy) for use with the TACS® 2 TdT Core Kit?Protocol Guideline
1. Fix tissue in 4% paraformaldehyde, 0.2% glutaraldehyde, 2% sucrose, 0.1 M cacodylate buffer, pH 7.3.

2. Wash 3-4 times in 0.1 M cacodylate buffer, pH 7.3 (recipe below).

3. Incubate for 30 minutes in 0.1 M cacodylate buffer containing 1% sucrose.

4. Wash 4 times in 0.1M cacodylate buffer.

5. Dehydrate in a graded ethanol series with 70% as the highest alcohol concentration.

6. Process LR White resin (G. Goping et al. (1999), J. Histochem. Cytochem. 44:289-295) followed by polymerization at 40°C in vacuum oven.

7. Section at 90-95 nm and collect onto copper grids.

8. Etch sections for 10 seconds in 100% ethanol.

9. Rinse 3 times in PBS for 10 minutes each.

10. (In situ Apoptosis Detection: perform washes in porcelain dishes. For incubations, it is convenient to spot the reaction mixes onto Parafilm and float the grid section side down on the “drop”.)

11. Immerse sample in 1X TdT Labeling Buffer (see Labeling Protocol) for 5 minutes.

12. Cover sample with 50 µL of Labeling Reaction Mix (see Labeling Protocol). Incubate for 60 minutes at 37°C in a humidity chamber.

13. Immerse sample in 1X TdT Stop Buffer (see Labeling Protocol) for 5 minutes.

14. Wash in dIH2O, 2 times for 2 minutes each.

15. Incubate sample in Streptavidin-Gold (e.g. at 2.5 µg/mL, 14-linker, from Eye Labs, Inc., San Mateo, CA) prepared in PBS + 1% BSA) overnight at 4°C. Note that the concentration of Streptavidin-Gold and the incubation time will depend on source and some empirical testing will be needed

16. Wash 4 times with PBS containing 1% BSA, 4 times in PBS, 3 times dH2O and stain for 20 minutes in uranyl acetate and 0.4 minutes with lead citrate.

Note: Use highest quality water available (e.g. double distilled) and ensure blades used for sectioning are very clean for optimum results. Omit secondary fixation with osmium tetroxide (OSO4) and/or membrane enhancement with potassium ferricyanide of the tissue. DNA end-labeling on osmicated cells was 50% less efficient than labeling on unosmicated cells.


1 M Cacodylate Buffer:


300 mM Tris-Cl pH 7.3

1 M sodium cacodylate

10 mM cobalt chloride


NOTE: This protocol requires the separate purchase of Streptavidin-Gold conjugate

HINTS: This kit has not been tested for use with resin embedded tissue. It should work with methacrylate.
4810-30-CK
TUNEL based kitsWhy do I lose my signal in a double labeling experiment with a TACS 2 TdT kit?

A paraffin embedded section was first stained using Vector Nova RED substrate kit (for peroxidase) to detect a cytoplase protein by immunohistochemistry. The section was then double labeled using TACS® 2 TDT Kit. After completing the TACS® protocol the red color stained by immunohistochemistry disappeared. What is the reason?
For double labeling experiments the TACS® protocol should be performed first. The conditions to permeabilize the sample (e.g. proteinase K) should be used to optimize antigen detection (i.e. antibody dilutions etc). The permeabilization step precedes the antibody detection step to avoid removal of antibody complexes.All TACS 2 TDT kits
BME and ECM ProteinsHow should BME be stored?BME should be stored at -80°C for optimal stability. Preparation of working aliquots is recommended. BME should be thawed overnight on ice at 4°C (refrigeration). Long term storage at 4°C is not recommended. Freeze/thaw cycles and gel-liquid phase transitions can compromise product integrity.3432-001-01, 3432-005-01,
3432-010-01,
3433-001-01, 3433-005-01,
3433-010-01,
3433-001-R1, 3433-005-R1,
3433-010-R1,
3434-001-02, 3434-005-02,
3445-001-01, 3445-005-01,
3445-010-01,
3532-001-02, 3532-005-02,
3532-010-02,
3533-001-02, 3533-005-02,
3533-010-02,
3632-001-02, 3632-005-02,
3632-010-02,
BME and ECM ProteinsCan BME be diluted?Yes, dilute BME in tissue culture media at physiological pH. BME will form a gel when diluted to 10 mg/ml; however further dilutions may require optimization. 3432-001-01, 3432-005-01,
3432-010-01,
3433-001-01, 3433-005-01,
3433-010-01,
3433-001-R1, 3433-005-R1,
3433-010-R1,
3434-001-02, 3434-005-02,
3445-001-01, 3445-005-01,
3445-010-01,
3532-001-02, 3532-005-02,
3532-010-02,
3533-001-02, 3533-005-02,
3533-010-02,
3632-001-02, 3632-005-02,
3632-010-02,
BME and ECM ProteinsWhat is BME and how does it work?Cultrex® Basement Membrane Extract (BME) matrix is purified from murine Engelbreth-Holm-Swarm (EHS) sarcoma. BME is composed of soluble basement membrane proteins that gel at 37°C to form a reconstituted basement membrane. Major components include laminin I, collagen IV, entactin, and heparin sulfate proteoglycan. BME can be used for promotion and maintenance of differentiated phenotype in a variety of cell types. The matrix may also be used as a barrier to assess the invasive potential of migrant cells. 3432-001-01, 3432-005-01,
3432-010-01,
3433-001-01, 3433-005-01,
3433-010-01,
3433-001-R1, 3433-005-R1,
3433-010-R1,
3434-001-02, 3434-005-02,
3445-001-01, 3445-005-01,
3445-010-01,
3532-001-02, 3532-005-02,
3532-010-02,
3533-001-02, 3533-005-02,
3533-010-02,
3632-001-02, 3632-005-02,
3632-010-02,
BME and ECM ProteinsWhat is the difference between BME and 3-D Culture Matrix ?Trevigen developed 3-D Culture Matrix to provide the most standardized basement membrane extract for use in 3-D Cultures. A special process is employed to reduce growth factors and provide material at a standard concentration. This material is then incorporated in a 3-D culture to validate efficacy. 3433-001-01, 3433-005-01,
3433-010-01,
3445-001-01, 3445-005-01,
3445-010-01
BME and ECM ProteinsWhat are 3-D cultures?3-D cultures are in vitro cultures where immortalized cell lines, primary cell lines, stem cells, or explants are placed within hydrogel matrices that mimic in vivo cell environments and allow cells to proliferate in 3 dimensions.3433-001-R1, 3433-005-R1,
3433-010-R1,
3445-001-01, 3445-005-01,
3445-010-01,
3532-001-02, 3532-005-02,
3532-010-02,
3533-001-02, 3533-005-02,
3533-010-02
BME and ECM ProteinsWhat is the advantage of 3-D culture over traditional 2-D culture?While 2-D culture has been used for studying many aspects of cell function and behavior, the tissue-culture treated plastic environment is unlike anything found within living organisms. As a result, cells in 2-D culture exhibit altered morphology, function, proliferation, and gene expression when compared to their emanating tissues. By placing these cells in a 3-D environment, they assume biological and biochemical characteristics similar to what is observed in vivo.3433-001-R1, 3433-005-R1,
3433-010-R1,
3445-001-01, 3445-005-01,
3445-010-01,
3532-001-02, 3532-005-02,
3532-010-02,
3533-001-02, 3533-005-02,
3533-010-02
BME and ECM ProteinsWhat are the variables associated with 3-D culture?The major variables associated with 3-D culture are cell type, cell seeding density, composition of hydrogel, thickness of hydrogel, stiffness of hydrogel, composition of cell culture medium, and time of culture.3433-001-R1, 3433-005-R1,
3433-010-R1,
3445-001-01, 3445-005-01,
3445-010-01,
3532-001-02, 3532-005-02,
3532-010-02,
3533-001-02, 3533-005-02,
3533-010-02
BME and ECM ProteinsWhat are the different types of 3-D culture?The two principal methods for conducting 3-D culture are the top assay and embedded assay. For the top assay, cells are seeded on a thick gel and a thin overlay is applied with the cell culture medium. For the embedded assay, cells are resuspended within a thick gel and the culture media is applied on top. The top assay is easier to setup, to control seeding densities, and to keep cells within one focal plane for analysis. 3433-001-R1, 3433-005-R1,
3433-010-R1,
3445-001-01, 3445-005-01,
3445-010-01,
3532-001-02, 3532-005-02,
3532-010-02,
3533-001-02, 3533-005-02,
3533-010-02
BME and ECM ProteinsWhich matrix should I use for 3-D culture?Choice of matrix should correspond to the environment that you wish to recapitulate. A basement membrane extract (BME) will recapitulate the basal lamina, which underlie most cells of epithelial or endothelial origin. Collagen I is the major constituent of connective tissue, and is commonly inhabited by stationary cells, such as fibrocytes and adipose cells, as well as migrating cells, such as mast cells, macrophages, monocytes, lymphocytes, plasma cells, and eosinophils.3433-001-R1, 3433-005-R1,
3433-010-R1,
3445-001-01, 3445-005-01,
3445-010-01,
3447-020-01,
3532-001-02, 3532-005-02,
3532-010-02,
3533-001-02, 3533-005-02,
3533-010-02
BME and ECM BME and ECM ProteinsHow should cells be cultured prior to setting up the 3-D culture?Cells need to be healthy and actively dividing in 2-D culture. Cells should be passaged two or three times after resuspension from cryopreservation, and they should never surpass 80% confluency during each passage. Cells should also be assessed using trypan blue, and they should exhibit less than 5% staining.3433-001-R1, 3433-005-R1,
3433-010-R1,
3445-001-01, 3445-005-01,
3445-010-01,
3447-020-01,
3532-001-02, 3532-005-02,
3532-010-02,
3533-001-02, 3533-005-02,
3533-010-02
BME and ECM ProteinsWhat type of analysis is typically applied to 3-D cultures?Within the cultures, cells may be assessed for morphology, apical/basal polarity, protein localization, and relative proliferation. In addition, cells may be isolated from the 3-D culture and evaluated for levels of RNA and protein expression, as well as modifications to DNA. 3433-001-R1, 3433-005-R1,
3433-010-R1,
3445-001-01, 3445-005-01,
3445-010-01,
3447-020-01,
3532-001-02, 3532-005-02,
3532-010-02,
3533-001-02, 3533-005-02,
3533-010-02
BME and ECM ProteinsHow would I measure the proliferation of my cells when cultured in a 3-D environment?Cellular proliferation in 3-D culture may be measured using the Cultrex® 3-D Culture Cell Proliferation Reagent.3445-096-01
BME and ECM ProteinsHow can I harvest my cells for subsequent analysis?Cells may be harvested from 3-D culture using the Cultrex® 3-D Culture Cell Harvesting Kit.3448-020-K
BME and ECM ProteinsAt what concentration should Laminin I be used?The recommended working concentration is 0.05-10 µg/cm 2 of growth surface (0.05 - 10 µg/ml) depending on cell type for thin coating; conditions must be optimized for each cell line or model. Laminin I should be used at 6 mg/ml for 3D culture applications.3400-010-01,
3400-010-02,
3400-010-03,
3446-005-01
BME and ECM ProteinsWhat is Laminin I?Laminin I is a major component of BME purified from murine EHS sarcoma. It is composed of α 1β1γ1 chains with a total MW of 800,000 Da. Laminin I increases cell adhesion, migration, growth, differentiation, neurite outgrowth, protease production, and malignancy. The response is dependent on cell type. 3400-010-01,
3400-010-02,
3400-010-03,
3446-005-01
BME and ECM ProteinsCan mouse Laminin I be used on glass cover slips?Coating glass cover slips with Laminin I is quite common and has been published extensively. 3400-010-01,
3400-010-02,
3400-010-03,
3446-005-01
BME and ECM ProteinsWhat is the difference between the Cultrex Collagen I (Rat) and the 3-D Collagen I Rat Tail?Our 3D Collagen has been tested extensively for the ability to promote growth and differentiation of cell types, visualized by morphology in three dimensions in vitro. The standard collagen undergoes the same basic purification and efficacy tests. The 3D collagen is the same as standard material; however it undergoes additional 3-D culture validation.3440-100-01,
3440-005-01, 3447-020-01
BME and ECM ProteinsWhat are the difference between Cultrex Collagen I and Cultrex Rat Collagen I (LV) Low Viscosity?Cultrex Rat Collagen I is provided at 5 mg/ml and is not pepsin treated, so it has intact telopeptides and is highly viscous. Cultrex Rat Collagen I (LV) Low Viscosity is provided at 3mg/mL, and it is easier to pipet. Both are provided in acetic acid and require pH neutralization prior to use. Please consult the provided product data sheets for pH neutralization instructions.
3440-100-01, 3440-005-01, 3447-020-01,
3443-100-01
Cultrex AssaysShould the wells used for the generating a standard curve be assayed using the same conditions and instrument settings as the assay plate?Yes. Identical conditions (time, temperature, volumes, etc.) should be used for generating standard curves to convert RFU to cell number. Also, identical instrument settings (gain, well read area, etc.) should be used for reading standard curve and assay plates. Black plates must be read from the top.3455-024-K,
3455-096-K,
3456-024-K,
3456-096-K, 3457-024-K,
3457-096-K,
3458-024-K,
3458-096-K,
3465-024-K,
3465-096-K,
3480-024-K,
3481-024-K,
3481-096-K,
3482-024-K,
3482-096-K,
3483-024-K,
3483-096-K,
3484-024-K,
3484-096-K
Cultrex AssaysWhat is cell migration?Cell migration is the movement of cells in response to a chemical stimulus; this is also known as chemotaxis. Cultrex® Cell Migration Assays evaluate cell migration based on the cells ability to traverse an uncoated membrane with 8 micron pores, in response to a chemotactic gradient. The cells must undergo cytoskeletal remodeling to fit into the pores and pull themselves through to the underside of the membrane. 3465-024-K, 3465-096-K
Cultrex AssaysWhat is cell invasion?Cell invasion is cell migration through a physiological barrier in response to a chemoattractant, and this recapitulates cell movement within a physiological environment which is composed of extracellular matrix proteins. Here, the membranes are coated with a layer of extracellular matrix proteins, and the cells must traverse this barrier through a combination of protein degradation and cellular locomotion. 3500-096-K, 3455-024-K, 3455-096-K, 3456-024-K, 3456-096-K, 3457-024-K, 3457-096-K, 3458-024-K, 3458-096-K, 3460-096-K
Cultrex AssaysWhat are the variables associated with cell invasion?The major variables associated with cell invasion are cell type, cell density, composition of the physiological barrier, thickness of the physiological barrier, chemoattractant that is used, and time of culture.3455-024-K,
3455-096-K,
3456-024-K,
3456-096-K, 3457-024-K,
3457-096-K,
3458-024-K,
3458-096-K,
3465-024-K,
3465-096-K,
3480-024-K,
3481-024-K,
3481-096-K,
3482-024-K,
3482-096-K,
3483-024-K,
3483-096-K,
3484-024-K,
3484-096-K,
3500-096-K
Cultrex AssaysWhich matrix should I use for cell invasion?Choice of matrix should correspond to the environment that you wish to recapitulate. Basement membrane extract (BME) will recreate the basal lamina, which underlie most cells of epithelial or endothelial origin. Collagen I is the major constituent of connective tissue, and is commonly inhabited by stationary cells, such as fibrocytes and adipose cells, as well as migrating cells, such as mast cells, macrophages, monocytes, lymphocytes, plasma cells, and eosinophils.3455-024-K,
3455-096-K,
3456-024-K,
3456-096-K, 3457-024-K,
3457-096-K,
3458-024-K,
3458-096-K,
3465-024-K,
3465-096-K,
3480-024-K,
3481-024-K,
3481-096-K,
3482-024-K,
3482-096-K,
3483-024-K,
3483-096-K,
3484-024-K,
3484-096-K,
Cultrex AssaysHow should cells be cultured prior to setting up the cell invasion assay?Cells need to be healthy and actively dividing in 2D culture. Cells should be passaged two or three times after resuspension from cryopreservation, and they should never surpass 80% confluency during each passage. Cells should also be assessed using trypan blue, and they should exhibit less than 5% staining.3455-024-K,
3455-096-K,
3456-024-K,
3456-096-K, 3457-024-K,
3457-096-K,
3458-024-K,
3458-096-K,
3465-024-K,
3465-096-K,
3480-024-K,
3481-024-K,
3481-096-K,
3482-024-K,
3482-096-K,
3483-024-K,
3483-096-K,
3484-024-K,
3484-096-K,
3500-096-K
Cultrex AssaysWill the Cultrex® Cell Invasion Assay work for my cells?The Cell invasion Assay is currently configured for invasive adherent cell lines. If your cell line is adherent and there is evidence in the scientific literature that your cell line is invasive in a Boyden chamber, it should be compatible with our assay. If this is unknown, then the invasive potential will need to be determined empirically. In general, cells that do not migrate in response to a chemoattractant will not divide. The variables listed in question 3 may need to be optimized to maximize the sensitivity of the assay.3455-024-K,
3455-096-K,
3456-024-K,
3456-096-K, 3457-024-K,
3457-096-K,
3458-024-K,
3458-096-K,
3465-024-K,
3465-096-K,
3480-024-K,
3481-024-K,
3481-096-K,
3482-024-K,
3482-096-K,
3483-024-K,
3483-096-K,
3484-024-K,
3484-096-K,
3500-096-K
Cultrex AssaysHow do the Trevigen Cell Invasion Assays compare to wound healing assays?Wound healing assays, also known as scratch assays, monitor cell migration laterally on a tissue culture treated plate. This is accomplished by generating a void in a cell monolayer by either removing cells or treating the surface of the plate to prevent cell growth in a designated area. The assay measures the ability of the cell monolayer to fill this void, and it may be conducted in the presence of extracellular matrix proteins. Since this assay is conducted within one chamber, there is no chemotactic gradient, and without the membrane, the cells are no longer required to change shape and squeeze through the pores. Another potential problem is that this assay does not control for differences in cell proliferation. While wound healing assays may be valuable for supplementing the Boyden chamber assay, it is not a replacement.3455-024-K,
3455-096-K,
3456-024-K,
3456-096-K, 3457-024-K,
3457-096-K,
3458-024-K,
3458-096-K,
3465-024-K,
3465-096-K,
3480-024-K,
3481-024-K,
3481-096-K,
3482-024-K,
3482-096-K,
3483-024-K,
3483-096-K,
3484-024-K,
3484-096-K,
3500-096-K
Cultrex AssaysCan the Calcein-labeled invasive cells be subcultured?Calcein AM cytoxicity should be determined empirically for each cell line or model. For best results, the cells should be removed from the cell dissociation solution and placed in fresh culture medium as soon as possible.3455-024-K,
3455-096-K,
3456-024-K,
3456-096-K, 3457-024-K,
3457-096-K,
3458-024-K,
3458-096-K,
3465-024-K,
3465-096-K,
3480-024-K,
3481-024-K,
3481-096-K,
3482-024-K,
3482-096-K,
3483-024-K,
3483-096-K,
3484-024-K,
3484-096-K,
3500-096-K
Cultrex AssaysWhat strains of mice has DIVAA™ been used with?DIVAA™ has been used with nude mice, the recommended and qualified strain for the assay. The mouse strain C57Bl has reportedly been used successfully with the kit. However, optimization is required, and variability is commonly reported. If you require C57BI mice for your experiments, it is recommended to allow the implantation to go for up to 15 days.3450-048-K, 3450-048-IK,
3450-048-SK
3450-048-K
Cultrex AssaysAre materials supplied in the DIVAA™ kit sterile?Yes, the materials supplied are sterile according to USP XXIV Chapter 71.3450-048-K, 3450-048-IK,
3450-048-SK
3450-048-K
Cultrex AssaysHow does DIVAA™ compare to the plug assay?The DIVAA™ angioreactor prevents assay errors due to re-adsorption of the basement membrane extract by the mouse. The assay also has lower dose requirements for angiogenic modulating factors compared to the plug assay, and multiple data points can be generated in one mouse.3450-048-K, 3450-048-IK,
3450-048-SK
3450-048-K
Cultrex AssaysWhat is DIVAA™?The Directed In Vivo Angiogenesis Assay (DIVAA™) is the first in vivo system for the study of angiogenesis that provides quantitative and reproducible results. Angioreactors, (silicone cylinders closed at one end) containing 20 µL; of basement membrane with/without angiogenic-modulating factors are implanted subcutaneously in the dorsal flank of nude mice. With the onset of angiogenesis, vascular endothelial cells proceed to grow into the basement membrane extract and form vessels in the angioreactor. As early as nine days post-implantation, there are enough cells to determine an effective dose response to angiogenic modulating factors. 3450-048-K, 3450-048-IK,
3450-048-SK
3450-048-K
Cultrex AssaysWhy does DIVAA™ contain a FGF/VEGF mixture to promote angiogenesis?While FGF-2 and VEGF have both been demonstrated to promote angiogenesis in DIVAA™, the FGF/VEGF mixture provides a synergistic effect allowing a drastic increase in response using less total growth factor. 3450-048-K, 3450-048-IK,
3450-048-SK
3450-048-K
Cultrex AssaysWhat is the difference between the FITC Lectin and FITC Dextran in the DIVAA protocol?FITC-Lectin specifically binds to endothelial cells, so it counts the total number of endothelial cells contained within the angioreactor. FITC-Dextran does not bind endothelial cells. Instead, it is dispersed within the blood of the mouse, being evenly distributed within the blood vessels, so it quantitates the total volume of blood contained within the vessels within the angioreactor. Both procedures have demonstrated equivalent results empirically.3450-048-K, 3450-048-IK,
3450-048-SK
3450-048-K
Cultrex AssaysWhere are the DIVAA™ angioreactors implanted?The angioreactors should be implanted on the dorsal lateral flanks. These areas exhibit sufficient vasculature to support initiation of angiogenesis, and they are also strategically positioned to prevent removal by the host mouse. Other areas may be used for implantation; however, this is outside of the product specification. Please consult the scientific literature for more information.3450-048-K, 3450-048-IK,
3450-048-SK
3450-048-K
Cultrex AssaysCan cells be placed within the angioreactor?While some groups have had success with implanting angioreactors containing viable cells, this application has not been qualified by Trevigen. Please consult the scientific literature for more information.3450-048-K, 3450-048-IK,
3450-048-SK
3450-048-K
Cultrex AssaysCan multiple conditions be employed in a single mouse?Using multiple conditions in the same mouse results in higher variability, so it is not recommended. It is hypothesized that angiogenesis modulating factors may be entering the blood stream and affecting angiogenesis in other locations within the animal.3450-048-K, 3450-048-IK,
3450-048-SK
3450-048-K
Cultrex AssaysWhat is the Tube Formation assay?The Tube Formation assay is based on the ability of endothelial cells to form three-dimensional capillary-like tubular structures when cultured on a hydrogel of reconstituted basement membrane extract (BME).3470-096-K
Cultrex AssaysWhat are the advantages of the Tube Formation assay?The Tube Formation assay is rapid, inexpensive and quantifiable. It can be used to identify potentially angiogenic and anti-angiogenic factors; to determine endothelial cell phenotype, and to study pathways and mechanisms involved in angiogenesis. It can be performed in a high throughput mode to screen for a large number of compounds. Therefore, the Tube Formation assay is the most widely used in vitro angiogenesis assay.3470-096-K
Cultrex AssaysWhat cell types can be used in the Tube Formation assay?The Tube Formation assay is specific for endothelial cells: either primary cells or immortalized cell lines. Only endothelial cells form capillary-like structures with a lumen inside, other cell types form other structures.3470-096-K
Cultrex AssaysWhat are the variables associated with the Tube Formation assay?The major variables associated with tube formation are composition of the BME hydrogel, thickness of hydrogel, cell density, composition of angiogenic factors in the assay medium, and assay period.3470-096-K
Cultrex AssaysWhich BME should I use for the Tube Formation assay?Reduced Growth Factor BME (RGF BME) is generally used for testing compounds that promote angiogenesis because formation of capillary-like structures (tubes) is significantly less, compared to complete basement membrane matrix (Normal BME). Trevigen®’s Tube Formation Kit includes a qualified production lot of RGF BME that exhibits reduced background tube formation in the absence of angiogenic factors.3470-096-K
Cultrex AssaysHow do I reduce spontaneous formation of tubular structures on BME gels in the absence of angiogenic factors?Primary endothelial cells, such as Human Umbilical Vein Endothelial Cells (HUVECs) form capillary-like structures in the absence of added angiogenic factors less often than immortalized endothelial cells. Titrate the number of cells and find optimal conditions for your specific cell line. When endothelial cells make fully-formed capillary structures in response to angiogenic activators, but not in their absence, you may proceed. Generally, reducing the number of cells per cm 2 of belled BME results in less background or spontaneous tube formation. 3470-096-K
Rspo1 Cell LineWhat kind of expression system was used to establish this cell line?
HA-R-Spondin1-Fc 293T cell line was developed originally by transfecting HEK293T cells with a plasmid containing the coding sequence of the murine R-Spondin1 to achieve stable expression. This protein has two terminal tags to improve purification and detection, one HA-tag on the amino terminal and a C-terminal murine IgG2a Fc fragment. There was no viral transduction involved in the development of this product.
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Rspo1 Cell LineWhat is the species of origin of the R-spondin1 expressed by this cell line? What is the specificity of this protein?
This is murine R-Spondin-1. The protein is conserved, and it is commonly used with human organoid culture as well. Conditioned medium from this cell line has been used extensively in the culture of human organoids by many labs across the world (Hans Clevers’ lab -Hubrecht Institute, Netherlands- being one of them).
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Rspo1 Cell LineFor how many passages can this cell line be grown?
This is a stably transfected immortalized cell line. We have passaged them under selection for more than 10 times without any issues. We would recommend building a tiered cell bank (opposed to serially passaging) for optimal stability over time.
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Rspo1 Cell LineHow can I detect the presence of R-spondin1 in the conditioned medium?
We recommend to perform a western blot to detect either R-spondin1, HA-tag or Fc as valid methods to confirm the expression of HA-R-spondin1-Fc. We suggest to test at least two different antibodies. The band detected should be at approximately 70-75 KDa.
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Rspo1 Cell LineIs there a functional assay to test the conditioned medium?
To determine how active the HA-R-spondin1-Fc conditioned medium or the purified protein is, we recommend performing a TopFlash assay. Compare HA-R-Spondin1-Fc conditioned medium to a source of R-spondin1 with known activity and, depending on the desired final concentration, decide how concentrated or diluted the conditioned medium is. We provide a TopFlash protocol on our website www.trevigen.com.
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Rspo1 Cell LineHow should R-Spondin1 conditioned medium be stored?
We recommend storing the HA-R-Spondin1-Fc conditioned medium frozen at -20°C for one month.
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