|72782S||1 Kit (100 assays)||
|Product Includes||Quantity (with Count)|
|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
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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