Flow cytometric analysis of target K562 cells (top row) or SUP-B15 cells (bottom row) labeled with CFSE, incubated with (right column) or without (left column) effector NK-92 cells at a 12:1 effector:target cell ratio for 4 h, and stained with 7-AAD for 15 min. Cells that stain positive for both 7-AAD and CFSE represent dying target cells and are located in Q2 of the dot plot.
|Product Includes||Quantity (with Count)||Solution Color|
|7-AAD Viability Dye||2 x 50 µl|
|CFSE Stock Solution||1 x 100 µl|
|Cell-Based Assay Buffer Tablet||1 x 4 ea|
NOTE: The recommended protocol labels cells with CFSE brightly enough to distinguish labeled and unlabeled cells, but dimly enough not to bleed into other channels. However, should brighter staining be desired, BSA can be omitted from the CFSE Staining Solution preparation.
Additional Reagents (Not Supplied):
NOTE: To properly analyze the data, the following control target cell groups are needed to set up the flow cytometer and compensation:
|Effector:Target Ratio||Effector Cell Suspension||Target Cell Suspension||Target Cell Medium||Final Volume|
|0||0 ml||1.5 ml||0 ml||1.5 ml|
|6.25:1||0.125 ml||1 ml||0.375 ml||1.5 ml|
|12.5:1||0.25 ml||1 ml||0.25 ml||1.5 ml|
|25:1||0.5 ml||1 ml||0 ml||1.5 ml|
NOTE: If additional surface markers are to be assayed, staining can be inserted at this point in the protocol.
|Problem||Possible Causes||Recommended Solutions|
|Low signal of CFSE||
|No difference in cytotoxicity among different effector cell to target cell ratios||
Posted February 2020
Protocol Id: 1964
The 7-AAD/CFSE Cell-Mediated Cytotoxicity Assay Kit employs CFSE (carboxyfluorescein succinimidyl ester) to label target cells within the mixed cell population and 7-AAD (7-aminoactinomycin D) to label dead cells. This kit provides an improvement over the traditional chromium-51 (51Cr) release assay to assess cell-mediated cytotoxicity. CFSE labeling is more sensitive, does not employ radioisotopes, and cytolysis can be assessed at the single-cell level using flow cytometry.
Cytolytic assays are routinely used to interrogate immune effector cell function. In order to interrogate immune effector cell cytolytic activity in a heterogeneous cell population of effector and target cells, it is imperative to be able to discriminate between effector and target cell populations with distinct phenotypes. As such, this kit is designed to label live target cells with the membrane permeable fluorescent dye CFSE prior to coculture with unlabelled effector cells, thus permitting a clear separation between live effector and target cells. After incubation of live effector and target cells, the DNA intercalating dye 7-AAD is added to label dying target cells with compromised plasma membranes. By using dyes with distinct spectral properties, a clear separation between four cell populations can be obtained using flow cytometry: live target cells, dead target cells, live effector cells, and dead effector cells (1).
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