CYTOTOXICITY AND APOPTOSIS DETECTION KITS ASSAYS

Quantify 4 populations of cells: 
live; early apoptotic; late apoptotic, and necrotic.

     In a single tube, scientists can now quantitate cytolytic activity using ICT's Total Cytotoxicity Test (TCT) kits. These kits do not use radioisotopes, so they are much faster, safer, and cheaper to use than ⁵¹Cr release assays. 
     These kits provide a direct measurement of cytotoxicity rather than using an indirect indicator, such as the release of LDH or ATP enzymes. ICT's TCT kit can further distinguish apoptosis from necrosis, leading to a more accurate assessment of cytolytic activity. At least 10% more cytolytic activity is often measured when apoptotic cells are also identified.
     ICT's TCT kit includes three fluorescent reagents. The first of these, the green CFSE membrane stain, is used to separate the effector cells from the target cells by staining all the target cells green. The second reagent, the red 7-AAD live/dead stain, measures cells killed by cytolytic activity by binding to the DNA of membrane- compromised cells. The kit can be used with just these 2 reagents (or you can use ICT's proprietary apoptosis reagent, SR-FLICA™, see below, for a more accurate reading, ICT's SR-FLICA™ reagent should be used: it is included in the kit). 
     Without using SR-FLICA™, The percentage of cytotoxicity is traditionally calculated by counting the number of dead target cells (which are stained both green and red, R2) and dividing by the total number of target cells (all green cells, R1 and R2).  This will give you a basic idea of the level of cell death. The following equation could be used:

     Early apoptotic cells induced by these same cytolytic effector cells will not, however be detected with just these 2 reagents. To address this problem, ICT's TCT kit offers a significant improvement compared to older methods: it includes an additional third reagent which measures apoptosis via detection of caspase activity (the orange/ red SR-FLICA™ poly caspases probe, SR-VAD-FMK). When this additional apoptotic activity is measured along with cytotoxic cell activity, you obtain a higher percentage of overall cell death.
     With this kit, all of the target cells are initially labeled with the green CFSE membrane stain, then the unstained effector cells are added. Following the incubation period, the orangey-red SR-FLICA™ reagent is added to label caspase-positive cells. Lastly, the red 7-AAD live/dead stain is added, thus staining all the necrotic dead cells red. 
     As only the target cells are stained green, and the effector cells are not, the two populations can be easily distinguished via FACS. Within the target cell population, only the dead target cells are stained red (7AAD), easily separating them from the green living target cells. 
     By adding SR-FLICA™, cells with active caspases are stained orangey-red. Some of the 7-AAD-positive cells will also dually stain with SR-FLICA™. These cells are actually in the late stages of apoptosis: they have active caspases, but are far enough along to also have permeabilized membranes, which makes them 7-AAD-positive.
     Cell that are SR-FLICA™-positive and 7-AAD-negative should be considered to be in the early stages of apoptosis: they have active caspases, but show no signs of necrosis yet. These cells are not detectable by any other method, and often account for over 10% of they dying population, making ICT's TCT assay far more accurate and reliable than any other method available. Try it today, catalog #971 or 972.

Order sheet

• Safe - does not use ⁵¹Cr radioisotopes 

• Easy - just wash the cells 
  and add the reagents to the media. 

• Accurate - early apoptotic cells can be detected for a true assessment of cell death; other assays can't detect them.

• Distinguish apoptosis from necrosis - other assays erroneously include late apoptotic cells as part of the necrotic population!

• Detect more cytolytic activity - use the SR-FLICA™ reagent to concurrently quantitate all apoptotic  cells. 

• Directly measure cytolytic activity - rather than a side-effect like enzyme release. 

• Quantitate 4 populations - analyze cells via flow cytometry. 

watch our cytotoxicity
  slide show

see data and protocols in our product release

see data in our  publication

see data in our general newsletter

read the detailed protocol in our total cytotoxicity manual

calculate the # cells needed 
with our spreadsheet

prices

Steps:
Stain target cells with CFSE
Add effector cells
Stain apoptotic cells with SR-FLICA™
Stain necrotic cells with 7AAD
Analyze via flow cytometry

Most samples will have a mixture of target cells (green ovals) stained with SR-FLICA™ (orange, apoptotic), and/or 7AAD (red, necrotic). All target cells will be stained with CFSE (green). Effector cells (purple stars) may also exhibit staining with SR-FLICA™ and 7AAD, but these cells will be excluded during FACS gating, as none of the effectors will be stained with CFSE (green), see Exclusion of effector cells, below. Control H is shown here as an example of the different populations (manual, p. 21). Calculate the # cells needed  with our spreadsheet.

Separation of target cells (green ovals, R3) from effector cells (purple stars, not green, left side) using FACS. As all target cells were initially stained green, the entire target cell population can be counted in R2+R1 (=R3). After the effector cells and red live/dead stain were added to the target cells, all the dead and late-apoptotic target cells were stained red, shifting them up the Y-axis (R2), clearly distinguishing them from the live target cells (R1). Older methods of assessing cytotoxicity (without using SR-FLICA™) would stop here. The percentage of cytotoxicity was traditionally calculated by counting the number of dead target cells (which are stained both green and red, R2) and dividing by the total number of target cells (all green cells, R1 and R2). This will give you a basic idea of cytotoxicity. 

Since ICT's TCT kit includes SR-FLICA™, you can get a far more accurate quantitation of cytotoxicity by including apoptotic cells.  Further analyze these cells by gating on R3 (left) and re-reading the samples (below).

After gating on R3, CFSE-green-stained target cells are further divided into 4 populations of cells:

1. Live cells
2. Early apoptotic cells - not detectable by any other methods!
3. Late apoptotic cells - often mistaken as necrotic cells!
4. Necrotic cells

Measure the true level of cytotoxicity! If the early apoptotic cells (7.98%) were not included in this calculation, the level of cytotoxicity would have been severely underestimated by 25%! 

See more pictures and data available in our manual, our publication, our product release, and in our newsletter.

calculate the # cells needed with our spreadsheet

Order sheet

Reference:
1. Olin, M. R., Choi, K. H., Lee, J., Molitor, T. 2005. Gamma delta t-lymphocyte cytotoxic activity against Mycobacterium bovis analyzed by flow cytometry. Journal of Immunological Methods vol 297.

SR-FLICA™ references:
1. Fiala, M., Lin, J., Ringman, J., Kermani-Arab, V., Tsao, G., Patel, A., Lossinsky, A.S., Graves, M.C. Gustavson, A., Sayre, J., Sofroni, E., Surarez, T., Chiappelli, F. and Bernard, G. 2005. Ineffective phagocytosis of amyloid-â by macrophages of Alzheimer’s disease patients. Journal of Alzheimer’s Disease vol 7:221-232. 
2. Graberek, J., L. Du, G.L. Johnson, B.W. Lee, D.J. Phelps and Z. Darzynkiewicz. 2002. Sequential activation of Caspases and serine proteases (serpases) during apoptosis. Cell Cycle vol 1:124-131. 
3. Osborne, M. K., Maxey, J. A., Bell, Wade E. 2004. Caspase inhibitors block entry into autogamy in Paramecium tetraurelia. Poster presented at the American Society for Microbiology, 2004. 
4. Tinner-Staines, B., VandenHoek, S. 2004. Using cryopreserved neuronal cells for multiwell neurotoxicity assays. Poster presented at the Society for Neuroscience October 2004 annual meeting.

Read all our references: pdf.

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