Réactifs et instruments pour l'immunologie, la biologie cellulaire et la biologie moléculaire.

Total Cytotoxicity Test BioAssay™ Kit

Imprimer

Référence 474046-125T

Conditionnement : 125Tests

Marque : US Biological

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474046 Total Cytotoxicity Test BioAssay™ Kit

Applications

Shipping Temp

Blue Ice

Storage Temp

4°C/-20°C

Sample Type:
Cell culture

Reagent Name:
7-AAD, CFSE, SR-VAD-FMK

Target:
Cell-mediated cytotoxicity, poly caspases

Excitation/Emission:
7-AAD–546nm/647nm; CFSE–492nm/520nm; SR-FLICA–565nm/600nm

Method of Analysis:
Flow cytometry

Intended Use:
Total Cytotoxicity & Apoptosis Assay is a single-tube, tri-color assay for quantitative assessment of cell-mediated cytolytic activity due to apoptosis and necrosis. The assay employs a green fluorescent cellular stain, CFSE, to label target cells, the red live/dead viability dye, 7-AAD, to identify the dead cells present in the cytotoxicity assay samples, and the orange-red SR-FLICA reagent, SR-VAD-FMK, to measure caspase activity in the target cell population. Analyze your results using a flow cytometer.

Test Principle:
Cytolytic activity is an important process for eliminating intracellular pathogens and cancer cells. Total Cytotoxicity BioAssay™ allows you to assess cytolytic activity in cell culture.

The Total Cytotoxicity & Apoptosis Assay includes three fluorescent reagents: CFSE, 7-AAD, and SR-FLICA. CFSE, a green fluorescing cellular stain, is utilized to identify the target cell population. The unstained effector cells are added and incubated with the target cells. SR-FLICA reagent is incubated with cells to identify those containing active caspases. 7-AAD, a red fluorescing live/dead stain, is then added to stain all necrotic cells red by binding to the DNA of membrane-compromised cells. Through flow cytometry, quantify four populations of cells: live, early apoptotic, late apoptotic, and necrotic.

Kit Components:
125 tests:
SR-FLICA Reagent (SR-VAD-FMK), 1 vial
CFSE target cell stain, 1 vial
7-AAD vital stain, 1 vial
10X Assay Buffer, 1x30mL

250 tests:
SR-FLICA Reagent (SR-VAD-FMK), 2 vials
CFSE target cell stain, 1 vial
7-AAD vital stain, 2 vials
10X Assay Buffer, 1x60mL

Storage and Stability:
Store powder at 4°C liquid at -20°C. Store other components at 4°C. Stable for 6 months For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.

Applications

Important Note: This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological. Toxicity and Hazards: All products should be handled by qualified personnel only, trained in laboratory procedures.

References

1. Lee-MacAry, A. E. et al. Development of a novel flow cytometric|cell-mediated cytotoxicity assay using the fluorophores PKH-26 and|TO-PRO-3 iodide. J Immunol Methods 252, 83-92 (2001).|2. Slezak, S. E. & Horan, P. K. Cell-mediated cytotoxicity. A highly sensitive|and informative flow cytometric assay. J Immunol Methods 117,|205-214 (1989).|3. Radosevic, K., Garritsen, H. S., Van Graft, M., De Grooth, B. G. & Greve,|J. A simple and sensitive flow cytometric assay for the determination|of the cytotoxic activity of human natural killer cells. J Immunol|Methods 135, 81-89 (1990).|4. Hatam, L., Schuval, S. & Bonagura, V. R. Flow cytometric analysis of|natural killer cell function as a clinical assay. Cytometry 16, 59-68,|doi:10.1002/cyto.990160109 (1994).|5. Lowdell, M. W. et al. The in vitro detection of anti-leukaemia-specific|cytotoxicity after autologous bone marrow transplantation for|acute leukaemia. Bone Marrow Transplant 19, 891-897, doi:10.1038/|sj.bmt.1700756 (1997).|6. O’Brien, M. C. & Bolton, W. E. Comparison of cell viability probes|compatible with fixation and permeabilization for combined surface|and intracellular staining in flow cytometry. Cytometry 19, 243-255,|doi:10.1002/cyto.990190308 (1995).|7. Van Hooijdonk, C. A., Glade, C. P. & Van Erp, P. E. TO-PRO-3 iodide: a|novel HeNe laser-excitable DNA stain as an alternative for propidium|iodide in multiparameter flow cytometry. Cytometry 17, 185-189,|doi:10.1002/cyto.990170212 (1994).|8. Olin, M. R., Hwa Choi, K., Lee, J. & Molitor, T. W. Gammadelta Tlymphocyte|cytotoxic activity against Mycobacterium bovis analyzed|by flow cytometry. J Immunol Methods 297, 1-11, doi:10.1016/j.|jim.2004.10.002 (2005).|9. Bedner, E., Smolewski, P., Amstad, P. & Darzynkiewicz, Z. Activation of|caspases measured in situ by binding of fluorochrome-labeled inhibitors|of caspases (FLICA): correlation with DNA fragmentation. Exp Cell|Res 259, 308-313, doi:10.1006/excr.2000.4955 (2000).