In many cell types the intracellular calcium (Ca2+) acts as a second messenges controlling important cell functions. Ca2+ responses are usually measured in vitro on cultured cells. Our lab has developed a model based on thick slices made from fresh tissues. By using fluorescent imaging microscopy it is possible to measure several functional responses of T cells after adding them onto fresh tissue slices. Depending on the timescale of the response, we have 3 standard experiments : - Intracellular Ca2+ as a proxy of cell activation - Localization, mobility and interaction with other cells - Cell death Here, I want to focus on the initial activation of T cells when they recognize their target in tissue slices. In our lab we focus on CAR-T cells in tumor slices. The advantage of the model is the intact environment with physiological expression of the target. The different steps of the protocol are the following: 1) The fresh tissue sample is embedded in agarose and cut in thick slices(400 µm). To make the workshop feasible, we will use tissue slices made from a tumor that has been frozen and thawed, or fixed tumor slices. 2) Immunostaining of the tissue slices (structural organization) and loading of immune cells with the calcium specific dye. 3) Loading of CAR-T cells on the slice directly beneath the microscope (upright multiphoton microscope) and the calcium responses of single cells are measured immediately and during the first 10-20 minutes after plating the cells. 4) Tracking of the loaded cells and extraction of the Ca2+ concentration in each T cell within the microscopic field over time (ImageJ or Icy followed by RStudio). a. We will distinguish different calcium profiles which encode information controlling cell activation. b. From these individual cell data, we can synchronize the responses and calculate the average Ca2+ responses. The main outputs are the maximal response, the Ca2+ level at a later timepoint and the percentage of responding ce