Apoptosis is a regulated process of cell death that occurs during embryonic development as well as maintenance of tissue homeostasis. The appearance of non-regulated apoptosis involves the possibility of different diseases, such as neurodegenerative diseases and cancer.
Understanding the mechanisms of programmed cell death or apoptosis, is an important aspect in the research of tissue homeostasis, tumor diseases, or the toxicity and efficacy of drugs, among others.
In this sense a wide number of methods dedicated to the identification of apoptotic cells and the analysis of the morphological, biochemical, and molecular processes that occur during this global biological process have been developed so far. Apoptotic cells are identified based on their fragmented DNA content and morphological changes including nuclear condensation, the phosphatidylserine flip-flop, and loss of mitochondrial membrane potential which can be detected by flow cytometry.
▌What exactly is apoptosis?
As we have already mentioned, apoptosis is a regulated process of cell death that occurs during embryonic development as well as maintenance of tissue homeostasis. In adults, apoptosis is used to remove cells that have been damaged beyond repair, homeostasis and immune system regulation.. Furthermore, inappropriately regulated apoptosis is implicated in different disease states, such as neurodegeneration disease and cancer.
Therefore, we can define apoptosis as a type of cell death in which a series of molecular steps in a cell lead to its death. This is one method the body uses to get rid of damaged cells.
▌Why is interesting to research about apoptosis?
Decreased or inhibited apoptosis is a feature of many malignancies, autoimmune disorders, and some viral infections.
Particularly, apoptosis plays an important role in a long list of diseases associated with cell survival, specially, in cancer, which has probably received the most research effort in this field.
Furthermore, there is an increased interest in research of apoptosis and its implications in other diseases such as AIDS, Alzheimer’s and other neurodegenerative diseases, Parkinson’s disease, and amyotrophic lateral sclerosis, as well as autoimmune disorders such as autoinmmune-hepatitis, and graft versus host disease.
► What makes apoptosis an important item in cancer research?
It has been demonstrated that there is a strong correlation in the regulation of apoptosis and some diseases, and this happens because when the balance between cell growth and cell death is not properly regulated, tissue and, subsequently, organ dysfunction occurs.
In this sense, apoptosis is not only a mechanism to control cell number and tissue size, mechanism known as homeostasis , but also to remove infected, damaged, or stressed cells from the body.
As we addressed before, apoptosis plays a very important role in cancer research, and this is because some cancers are triggered by viral inhibition of apoptosis and apoptosis blocking is the is the main mechanism of resistance to chemotherapy which makes apoptosis a key target for cancer research. Therefore, understanding of the apoptosis machinery and resistance to apoptosis is a key field of research in order to fight against this disease.
Specific induction of apoptosis or blocking of inhibitory mechanisms have been indicated as therapeutic targets and, in this sense, various types of cancers could be diagnosed because of defects in the apoptosis mechanism.
▌Apoptotic cell-derived exosomes (extracellular versicles):
Exosomes are small (~40‐150 nm) extracellular vesicles (EVs) released from all cell types and found in body fluids and cell culture supernatants and they have been proposed to provide means for intercellular exchange of macromolecules, allowing the transfer of proteins, lipids, mRNA and miRNA, contributing to intercellular communication in relevant biological processes, including apoptosis, among others.
In this sense, like healthy live cells, apoptotic cells can also release extracellular vesicles (apoptotic extracellular vesicles) and with the development of detection technology, researchers have found these smaller vesicles produced by dying cells in addition to traditional apoptotic bodies very interesting for apoptosis research.
Furthermore, several recent studies have concluded that apoptotic exosomes exist and play a crucial role in various pathological and physiological conditions in humans.
In this regard, future research work in this field will probably be focusing on human diseases whose etiologies are implicated in chronic inflammation or immune responses, such as cancers, chronic allergies, autoimmune diseases, and degenerative neuroskeletal and musculoskeletal diseases.
Discover more about exosomes and their applications by visiting our exosome website or contact us for further information.
▌Annexin V staining is a common method used in order to detect apoptotic cells, but why is this?
Firstly, we have to mention that the surface of healthy cells is characterized by lipids that are distributed asymmetrically on the inner and outer leaflet of the plasma membrane.
In this sense, phosphatidylserine (PS), which is one of these lipids, is normally confined to the inner leaflet of the plasma membrane and is only exposed to the cytoplasm. During apoptosis, though, PS is exposed on the outer leaflet of the plasma membrane. This phenomenon is known as PS flip-flop.
This is the reason why it Annexin V is used to detect apoptotic cells, because this annexin V, a 36 kDa calcium-binding protein, is able to bind specifically to PS, therefore, fluorescently labeled annexin V can be used to target PS that is externally exposed on apoptotic cells by flow cytometry.
▌Annexin V binds to Phosphatidylserine (PS) allowing apoptosis detection by flow cytometry
Exposure of PS on the external surface of the cell membrane has been reported in apoptotic cells, this occurs in the early phases of apoptotic cell death during which the cell membrane remains intact. In leukocyte apoptosis, PS on the outer surface of the cell marks the cell for recognition and phagocytosis by macrophages.
In this sense, the human vascular anticoagulant, annexin V, is a 35-36 kDa Ca2+ dependent phospholipids binding protein that has a high affinity for PS, and shows minimal binding to phosphatidylcholine and sphingomyelin.
Changes in PS asymmetry, which is analyzed by measuring annexin V binding to the cell membrane, were detected before morphological changes associated with apoptosis have occurred and before membrane integrity has been lost.
▌Different annexin V Staining apoptotic Assays:
Immunostep could see the opportunity of building a perfect tool to identify and quantify apoptotic cells on a single cell basis by flow cytometry and we launched a line of assays for apoptotic detection. The main characteristics of these line of products for apoptosis are:
► They are a highly sensitive assays to detect early to late apoptotic stages
► We built a large number of assays to detect different parameters
► We validated the assays by flow cytometry with many different type of cells and also with many different types of drugs
► We offer different fluorescent options for multiple laser excitation sources
For example, Annexin V labelled with PE (red fluorescence) can identify and quantitate apoptotic cells on a single-cell basis by flow cytometry. Staining cells simultaneously with Annexin V– PE and the non-vital 7-Amino-Actinomycin D (far red fluorescence) allows (bivariate analysis) the discrimination of intact cells (Annexin V-PE negative, 7-AAD negative), early apoptotic (Annexin V-PE positive, 7-AAD negative) and late apoptotic or necrotic cells (Annexin V-PE positive, 7-AAD positive).
On the other hand, Annexin V labelled with FITC (green fluorescence) can identify and quantitate apoptotic cells on a single-cell basis by flow cytometry. Staining cells simultaneously with Annexin V –FITC and the non-vital dye propidium iodide (red fluorescence) allows (bivariate analysis) the discrimination of intact cells (Annexin V-FITC negative, PI negative), early apoptotic (Annexin V-FITC positive, PI negative) and late apoptotic or necrotic cells ( Annexin V-FITC positive, PI positive
▌Example protocol for annexin v expression in apoptotic peripheral blood lymphocytes
Here we are going to describe an example protocol for annexing V Expression in apoptotic peripheral blood lymphocytes in order to show how this is a very easy and fast assay to perform in order to detected apoptotic cells. Please contact us for any additional question or request further information using our contact form.
- MN-Cells (Mononuclear cells) are separated by Ficoll, from peripheral blood.
- Apoptosis induction in leukocytes are incubating for 6 hours with H2O2 200µM.
- 1 million cells are harvested after the apoptosis induction. The supernatant is removed by centrifugation.
- Them, 100uL of PBS is added and 20uL of the CD19 APC (Ref. CD19APCA3-B1 – IMMUNOSTEP) and it is incubated during 15 min.
- The cells are washed once with temperate PBS and then, the cells are resuspended in 1 X Annexin-binding buffer 0,5 ml.
- After that 5 μl of the Annexin V-FITC is added and 5 μl of PI, to each 100 µl of cell suspension (up 1×105 cells).
- The cells are incubated for 15 minutes at room temperature, and then, they can be analyzed by flow cytometry.
So far, Immunostep annexin related products have accumulated more than 300 citations in pubmed. Find out more about these and other reagent kits for apoptotic cells detection on our website, or contact us for further information, we are looking forward to hearing about your research needs.
► References:
- https://www.pnas.org/doi/10.1073/pnas.94.24.12736
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1120576/
- Koopman, G., Reutelingsperger, C. P., Kuijten, G. A. M., Keehnen, R. M. J., Pals, S. T., and van Oers, M. H. J. 1994. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84: 1415.
- http://cellanalysis.imaisd.es/wp-content/uploads/2022/04/TDS-ANXVKF_EN.pdf
- http://cellanalysis.imaisd.es/wp-content/uploads/2022/04/TDS-ANXVKPE_EN.pdf
- https://pubmed.ncbi.nlm.nih.gov/31915368/#:~:text=Apoptosis%2C%20a%20type%20of%20programmed,is%20not%20yet%20commonly%20accepted.
