Vybrant Apoptosis Assay Kit #3 From Molecular Probes (Invitrogen)

Apoptosis is a tightly regulated process of cell death which is crucial for normal development. This process is frequently deregulated in human pathologies such as cancer. Apoptosis has a number of morphological and biochemical characteristics which enable it to be distinguished from other types of cell death such as necrosis. These characteristics include nuclear chromatin fragmentation and upregulation of proteins such as caspases.

The Vybrant Apoptosis Kit #3 is from a range of apoptosis assay kits from Molecular Probes (Invitrogen) which measure apoptosis through different chemical and morphological characteristics. This kit contains the stains propidium iodide and FITC Annexin V, which enable a researcher to distinguish live, early apoptotic and late apoptotic/dead cells within a single sample. Propidium iodide is a ‘vital’ dye, which is impermeant to live cells, but is able to enter dead cells and bind tightly to DNA. This results in a red fluorescence. Annexin V is a calcium-dependent phospholipid-binding protein which binds with high affinity to the membrane phospholipid, phosphatidylserine (PS). In healthy cells, PS is located on the inner leaflet of the plasma membrane, but in one of the earliest steps of apoptosis, this phospholipid is translocated to the outer leaflet, exposing it to binding by exogenously added Annexin V. This kit contains Annexin V conjugated to the fluorescent molecule FITC, which can easily be distinguished from PI fluorescence using a 488 nm argon laser, in either flow cytometry or microscopy applications.

Sample preparation for flow cytometry using this kit is simple, with either adherent or cells in suspension culture. After harvesting, cells are washed with ice cold phosphate buffered saline (PBS), centrifuged and resuspended in an annexin binding buffer (provided in the kit as a 5x concentrate). At this stage, the cells should be counted and the cell concentration adjusted to ~1 x 10⁶ cells/ml. Staining of the cells involves addition of 5 ul of annexin V-FITC and 1 ul of PI (100 mg/ml stock provided, needs to be diluted to 100 ug/ml) and incubation at room temperature for 15 minutes. After this period, the samples should be kept on ice and measured as soon as possible.

I have used this kit on adherent mammary carcinoma cell lines (MCF-7 and MDA-MB-231) and have found the assay to be easy to set up and reliable. The experiments in which I have used this kit involved the treatment of stable mammary carcinoma cell lines with chemotherapy drugs with or without serum with analysis after 24 hours of treatment. I was initially concerned about using an Annexin-V stain on adherent cells harvested through trysinization because of the possible degradation of surface PS, but have not found this to be a problem and have seen distinct cell populations within my samples.

In summary, this kit is extremely user friendly, reliable and effective and I certainly recommend it. The only drawback of the kit is the expense due to the recombinant Annexin V-FITC and hence experiments should be planned carefully prior to use.