- What is R&D Systems doing to reduce its use of packaging from non-renewable resources?
- Environmental stewardship is important to R&D Systems and its employees. R&D Systems is ISO 14001:2015 Certified and as part of this we are continuously reviewing our sustainability practices and "green" options. First and foremost, the energy expended to ship back the styrofoam box is more detrimental to the environment than having the facility re-use or recycle it. Our stance is to encourage our customers to implement a recycling program locally. At R&D Systems we have chosen to reduce the use of styrofoam as much as possible by: doing extensive stability testing in order to determine which products can be shipped with minimal packaging at ambient temperatures, converting the use of non-recyclable packaging materials to recyclable plastics, cardboard, or biodegradable materials, and continuing to investigate alternatives to dry ice shipments, the use of re-usable containers, and gel packs that allow for smaller styrofoam containers. Employees were key to initiating a recycling program at our facilities. Internally, we recycle paper, plastic, cardboard, aluminum, and glass.
Annexin V 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.
- I am studying apoptosis in adherent cells. Can I trypsinize the cells when using Annexin V-FITC to stain the cells?
- Adherent cells undergoing apoptosis automatically detach from the plate and may not require trypsinization. If there are attached cells, they may be trypsinized without affecting phosphatidylserine (PS).
- I do not have access to flow cytometer in my building. Can I stain for Annexin V and propidium iodide (PI), fix the cells, then analyze them in a different facility?
- Fixing the cells can affect cell permeability, enhancing the uptake of Annexin V and PI intracellularly. As a result, the profile of fixed cells may be different from unfixed cells, and the use of appropriate controls is recommended.
- 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.
Caspase Activity Assays
- 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.
Phosphatase Activity Assays
- What is R&D Systems doing to reduce its use of packaging from non-renewable resources?
- Environmental stewardship is important to R&D Systems and its employees. R&D Systems is ISO 14001:2015 Certified and as part of this we are continuously reviewing our sustainability practices and "green" options. First and foremost, the energy expended to ship back the styrofoam box is more detrimental to the environment than having the facility re-use or recycle it. Our stance is to encourage our customers to implement a recycling program locally. At R&D Systems we have chosen to reduce the use of styrofoam as much as possible by: doing extensive stability testing in order to determine which products can be shipped with minimal packaging at ambient temperatures, converting the use of non-recyclable packaging materials to recyclable plastics, cardboard, or biodegradable materials, and continuing to investigate alternatives to dry ice shipments, the use of re-usable containers, and gel packs that allow for smaller styrofoam containers. Employees were key to initiating a recycling program at our facilities. Internally, we recycle paper, plastic, cardboard, aluminum, and glass.
TUNEL Assays
- 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.