Flow Cytometry: Annexin V Apoptosis Kit [FITC] [NBP2-29373] - Controls run before the experimental samples are analyzed, to adjust the fluorescent compensation on the flow cytometer due to the FITC signal from the ...read more
Flow Cytometry: Annexin V Apoptosis Kit [FITC] [NBP2-29373] - Adherent RAW cells treated with actinomycin-D, detached with a cell detachment solution and stained with the Annexin V Apoptosis Kit [FITC].
Annexin V Apoptosis Kit [FITC] can identify apoptosis at an earlier stage than kits based on DNA fragmentation in the nucleus. However, it is like most assays and has limitations. Since Annexin V staining precedes the loss of membrane integrity which accompanies the later stages of cell death resulting from either apoptotic or necrotic processes, staining with Annexin V-FITC is typically used in conjunction with a live/dead dye such as propidium iodide (PI) or 7-Amino-Actinomycin (7-AAD) to allow the investigator to identify early apoptotic cells (PI negative, Annexin V-FITC positive) from dead cells (PI positive, AnnexinV-FITC positive). Viable cells with intact membranes exclude PI, whereas the membranes of dead and damaged cells are permeable to PI. For this reason, Annexin V staining has to be performed on live cells as opposed to assays which require para- formaldehyde/ethanol fixed cells.
The Annexin V assay works in the following manner: Cells that are viable are both Annexin V-FITC and PI negative. While cells that are in early apoptosis are Annexin V-FITC positive and PI negative and cells that are in late apoptosis or already dead are both FITC Annexin V and PI positive. This assay does not distinguish between cells that have undergone apoptotic death versus those that have died as a result of a necrotic pathway because the dead cells will stain with both Annexin V-FITC and PI (see image). However, when apoptosis is measured over time, cells can be often tracked from Annexin V- FITC and PI negative (viable, or no measurable apoptosis), to Annexin V-FITC positive and PI negative (early apoptosis, membrane integrity is present) and finally to Annexin V- FITC and PI positive (end stage apoptosis and death). The movement of cells through these three stages suggests apoptosis. In contrast, a single observation indicating that cells are both Annexin V-FITC and PI positive, in of itself, reveals less information about the process by which the cells underwent their demise. For this reason, it is a good idea to analyze samples from multiple time points.
Kit Type
Apoptosis Kit
Gene
ANXA5
Applications/Dilutions
Application Notes
Use in Flow cell surface reported in scientific literature (PMID 23955790) Use the Control Cells provided to set up compensation and quadrants. The Control Cells are positive for both Annexin V-FITC and PI. The basal level of apoptosis and necrosis varies considerably within a give cell population. Even in the absence of induced apoptosis, most cell populations will contain a minor percentage of cells that are positive for apoptosis ((Annexin V-FITC positive, PI negative) and dead, necrotic, or in the late stages of apoptosis (Annexin V-FITC positive, PI positive). Thus an untreated cell population is used to define the basal level of apoptotic and dead cells. Determine the percentage of cells that have been induced to undergo apoptosis by subtracting the percentage of apoptotic cells in the untreated from the treated population. Since cell death is the eventual outcome of cells undergoing apoptosis, cells in the late stages of apoptosis will have a damaged membrane and stain positive for PI as well as for Annexin V-FITC. Consequently cells which have undergone necrosis are not distinguishable from those which have undergone apoptosis. Use in Immunocytochemistry/immunofluorescence reported in scientific literature (PMID 25947082). Use in FLOW cytometry reported in scientific literature ( PMID 27448441)
Reviewed Applications
Read 2 Reviews rated 4 using NBP2-29373 in the following application:
Human reactivity reported in scientific literature (PMID: 23955790) Mouse reactivity reported in scientific literature (PMID: 22566271). Rat reactivity reported in scientific literature (PMID: 25947082)
Packaging, Storage & Formulations
Storage
Store at 4C. Do not freeze.
Kit Components
Components
Propidium Iodide (PI) Solution
Annexin V-FITC
10X PBS Solution
10X Binding Buffer
Compensation Control Cells
Notes
ADDITIONAL ITEMS REQUIRED (NOT INCLUDED IN THE KIT) Distilled H2O Flow Cytometer Experimental Cells Centrifuge Positive Control Cell Line (recommended, not required)
Alternate Names for Annexin V Apoptosis Kit [FITC]
Anchorin CII
annexin A5
Annexin V
annexin-5
ANX5
ANXA5
ANXV
Calphobindin I
Endonexin II
ENX2CBP-I
Lipocortin V
PAP-I
Placental anticoagulant protein 4
Placental anticoagulant protein I
PP4
Thromboplastin inhibitor
VAC-alpha
Vascular anticoagulant-alpha
Background
Apoptosis is the term that describes programmed cell death. It is believed to take place in the majority of animal and plant cells. It is a distinct event that triggers characteristic morphological and biological changes in the cellular life cycle. One of the early events that happens in the apoptotic pathway is the loss of plasma membrane asymmetry. In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular environment. Annexin V when labeled with a fluorescent tag, such as FITC, can be used with flow cytometry to measure this event. Annexin V is a 35-36 kDa Ca2+ dependent phospholipid-binding protein that has a high affinity for PS, and binds to cells with exposed PS.
Limitations
This product is for research use only and is not approved for use in humans or in clinical diagnosis. Kits are guaranteed for 6 months from date of receipt.
Chiavari M, Ciotti G, Canonico F et al. PDIA3 Reduce Glioma-associated Macrophage/microglia Pro-tumor Activation trough IL-6-STAT3-PDIA3 Pathway. Research Square Aug 4 2020
Skivka LM, Fedorchuk OG, Bezdeneznykh NO. The effect of antineoplastic drug NSC631570 on immunogenicity of B16 melanoma. J Exp Integr Med 2014 Mar 24 (Flow-CS, Mouse)
Bioinformatics Tool for Annexin V Kit (NBP2-29373)
Discover related pathways, diseases and genes to Annexin V Kit (NBP2-29373). Need help?
Read the Bioinformatics Tool Guide for instructions on using this tool.
Diseases for Annexin V Kit (NBP2-29373)
Discover more about diseases related to Annexin V Kit (NBP2-29373).
PRODUCT AVAILABILITY: Update Regarding the Evolving COVID-19 Situation
Bio-Techne appreciates the critical role that you and our products and services play in research efforts to further scientific innovation and discovery. We are continually assessing our manufacturing and supplier capabilities during the COVID-19 situation and are implementing precautionary measures to ensure uninterrupted supply of products and services. Currently, and as we abide by local shelter in place orders across the world, we are fully operational and do not anticipate any material supply disruptions across our Bio-Techne brands and product lines. As the situation evolves, our goal is to utilize preventive measures to reduce the threat that COVID-19 poses to our ability to meet the needs of our customers globally.