- Additional information
- ASSAY PRINCIPLE
- Readable Documents
- Kit contents and storage
- No radioactive materials required.
- Applications – Flow cytometry or Fluorescent microscopy.
- Detects cytolytic activity at a cellular level.
- Works with multiple types of mammalian cell lines.
The most commonly used method to measure CMC/ADCC is a radioactive chromium-51 (51Cr) release assays (2). There are several disadvantages with this assay: it is expensive, difficult to load certain cell types, expensive to dispose of due to strict environmental regulations, and has high background from spontaneous release of 51Cr. With the use of flow cytometry, it is now possible to eliminate the need for radioactive material and increase the ability to quantify cytolytic activity on a single cell bases. Various groups have demonstrated that measuring CMC/ADCC activity by flow cytometry has a strong (95%) correlation with the traditional 51Cr release assay (3,4,5,6).
A cell tracking dye CFSE ( 7,8,9) is utilized to label the target cell population. After the assay has run its experimental protocol, 7AAD (live/dead) (10,11) is added to measure cell death. 7AAD only enters membrane-compromised cells and binds to DNA.
Flow cytometry is utilized to gate on the target cells and measure 7AAD negative vs 7AAD postitve cells. % cytotoxicity is calculated by the following equation (see experimental example below):
7AAD positive (upper right quadrant)=R1/ 7AAD Postitve (upper right quadrant)= R1 + 7AAD negative (lower right quadrant)=R2 x100
To test natural killer ability of swine gd lymphocytes, K562 cells were stained and adjusted to a final concentration of 1 X 104 cells/100 ul PRMI containing 10 % FBS. gd lymphocytes were added at E/T ratios of 25:1, 50:1, and 100:1 and adjusted to a total volume of 400 ul RPMI, then incubated for 4 hours at 37º C in a sterile capped facs tube. Following incubation live/dead stain was added directly to each tube, incubated for 15 min on ice and analyzed by flow cytometry.
Kit contents and storage
|Part # 4002||CFSE Membrane Stain, 4 Vials||2-8°C|
|Part # 4003||7AAD Live/Dead Stain, 3 Vials||2-8°C|
|a. Perussia, B., (1998). Current Topics in Microbiology and Immunology 230, p63.
b. Whiteside, T.L., Rinaldo, C.R. and Herberman, R.B. (1992) Cytolytic Cellfunctions. In: N.R. Rose, E.C. de Macario (Eds.), Manual of Clinical Laboratory Immunology. American Society for Microbiology. Washington, DC, p. 220.
|Brunner, K.T., Manuel, J., Cerotini, J.C., Chapuis, B., (1968). Quantitative Assay of the Lytic Action of Immune Lymphoid Cells on Cr-labelled Allogenic Target Cells in- vitro; Inhibition by Iso-antibody and by Drugs, Immunology 14,181.|
|Lee-MacAry, A.E., Ross, E.L, Davies, D., and Wilkinson, R.W., (2001). Development of a Novel Flow Cytometric Cell-mediated Cytotoxcity Assay Using the Fluorophores PKH-26 and TO-PRO-3 Iodide. J. Immunology. Met 252, 83-92.|
|Gogoy-Ramirez, K., Franck, K., and Gains, H., (2000). A Novel Method for the Simultaneous Assessment of Natural Killer Cell Conjugate Formation and Cytotoxicity at the Single-cell Level by Multi-parameter Flow Cytometry. Journal of Immunology. Met 239, 35-44.|
|Goldberg, J.E., Sherwood, S.W., Clayberger, C., (1999). A Novel Method for Measuring CTL and NK Cell-mediated Cytotoxicity Using Annexin V and Two-color Flow Cytometry. Journal of Immunology. Methods 224, 1.|
|Hatam, L,. Schuval, S., Bonagura, V.R., (1994). Flow Cytometric Analysis of Natural Killer Cell Function as a Clinical Assay. Cytometry 16,59.|
|L.S De Clerck et al.,J. Immunol. Meth. 172, 115 (1994).|
|M. Bronner-Fraser,J. Cell Biol. 101, 610 (1985).|
|Rabinovitch, P.S., et al., J. Immunol. 136, 2769 (1986).|
|Su, X.,J. Immunol. 156, 156, 4198 (1996).|
|Olin, MR. Lee, J. Choi, K, and Molitor, Tw. gd T-lymphocyte Cytotoxic Activity against Mycobaterium bovis Analysed by Flow Cytometry: Journal of Immunological Methods; Publication in process.|
|Olin, MR. Thesis, K. Cho, J. and Molitor, T.W. Morphine Suppresses Microglial Directed Cytolytic Activity by gd Lymphocytes. Journal of Neuroimmunology; Publication in process.|
|Tetanus toxoid provides efficient T-cell help for the induction of HA-1H cytotoxic T cells||http://www.blackwell-synergy.com/doi/abs/10.1111/j.1537-2995.2006.00872.x||Britta EizVesper, Peter A. Horn, Claudia Daubert, Barbara Khattab, and Rainer Blasczyk||Transplantation and Cellular Engineering||2006|
|Immunological and clinical profile of adult patients with selective immunoglobulin subclass deficiency: response to intravenous immunoglobulin therapy||http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2249.2009.04062.x/full||F. Abrahamian, S. Agrawal, S. Gupta||Clinical & Experimental Immunology||Vol 159, Issue 3, March 2010|
|Distinct Roles for the NK Cell-Activating Receptors in Mediating Interactions with Dendritic Cells and Tumor Cells||http://www.jimmunol.org/content/186/1/222.abstract||Lu-En Wai, Jordan A. Garcia, Olivia M. Martinez and Sheri M. Krams||The Journal of Immunology||January 1, 2011, vol. 186 no. 1 222-229|