In this on-demand SelectScience webinar, Dr. Anne Lodge, Chief Science and Innovation Officer at Cellero, discusses how the Incucyte® Live-Cell Analysis offers walkaway monitoring of tumor cell lysis using fluorescent labels of cytotoxicity. Lodge presents data using tumor antigen-specific T cells from Cellero and widely available tumor cell lines to demonstrate specific lysis of tumor cells. Important technical factors are also discussed.
Read on for highlights of the live Q&A session or register to watch the webinar at any time that suits you.
Q: Do you use preactivated effectors or naive?
AL: The data shown in the presentation is with cells that we have thawed and immediately placed in the assay. NK cells are completely naive and for the T cells, I would consider preactivated.
AL: They are not necessarily inconsistent, but they reveal something about what is going on in the culture. For example, there could be more cells, if there’s a comparison between different target cell suspensions, or there is a difference in the cell size area.
AL: The use of non-specific effectors is important because we do see non-specific killing. The addition of the appropriate antigenic peptide can also reveal specific killing, especially when there isn't killing in the absence of peptide.
AL: You will be trying to see cell killing and be sure it is specific. That is difficult but I would run some allogeneic cells as controls. Those might give you an idea of non-specific killing.
AL: Yes, you would add the Annexin V at the start of the assay. Sartorius recommends the caspase reagent for adherent cells though.
AL: No, we're still demonstrating specificity with appropriate targets, but I can tell you that our Her2 specific T cells are cytotoxic against appropriate targets, as are the NY-ESO T cells.
AL: The assay using the Incucyte® uses fewer cells and needs less hands-on time.
AL: Yes, there are other reagents that can be used for labeling, particularly for Annexin V. We haven’t tried alternatives because we wanted to limit the variables as we worked out the assay.
AL: They can be expanded but we do not support that because it has been difficult for people to reproduce our expansion protocol.
AL: There is no minimum purity, we see good cytotoxicity when there is just 10% of the specific effectors present.
AL: That's the problem with that method. If T cells are adsorbed (and they are) then they contribute to signal and sort of drown out the loss of signal from target cells.
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