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
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.
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
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.
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.
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.
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.
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.
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)
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.