IncuCyte® Immune Cell Killing FAQs

Immuno-Oncology

Yes, as long as the fluorophore excitation and emission spectra are compatible with the IncuCyte® fluorescence imaging channels (green channel: excitation 440–480 nm, emission 504–544 nm; red channel: excitation 565–605 nm, emission 625–705 nm). Note that data quality may be compromised if fluorophore expression is unstable over time or results in heterogeneous labeling. We recommend our IncuCyte® NucLight Red or Green Lentiviral Reagents, as they are ideally suited for long-term live-cell studies. The NucLight labeling reagents do not disturb cell health and are expressed stably and homogeneously.

Yes. Please see our protocol for suspension target cell lines.

No. Cytoplasmic cell labels (e.g., IncuCyte® CytoLight Red or Green Lentivirus Reagents) can be used to label target cells; however, you will not be able to resolve and count individual target cells unless you have the Incucyte® Cell-by-Cell Software Analysis Module installed. Instead, cell area metrics such as Object Area or Confluence would be used to measure proliferation.

There is no need to pre-label your target cells when using adherent tumor cells. Tumor cell death is detected using either the IncuCyte® Caspase-3/7 Apoptosis Reagent (Cat # 4440 or 4704), or IncuCyte® Annexin V Apoptosis Reagent (Cat # 4641 or 4642) which is simply added to your assay along with your treatments. Although there is no need to label your target cells, you will find that using our IncuCyte NucLight Lentivirus Reagents to label your tumor cells will enable you to make simultaneous measurements of viable target cell count in real time. If you are using suspension target cells, they will need to be prelabeled. Measurements of non-adherent tumor cell killing are made by quantifying the reduction in target-cell fluorescence over time.

Yes. We have successfully performed immune cell killing experiments in a 384-well format.

Yes.  When measuring non-adherent target cell killing the Cell by Cell analysis software enables the user to quantify both features of the labelled target cell population and the non-labelled effector cell population (proliferation and death).  With adherent target cells the software enables the user to track effector cell populations in the presence of target cells.

Yes. Simply harvest cells/supernatants when the assay is complete, and perform your choice of further analysis.  Workflow protocols exist for use of this assay in combination with iQue T cell activation kit (TCA) to capture cytokine changes and activation states of effector cells.

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