- 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.