An Imaging Technique to Study the Calcium Response in Bacteria-Infected Cells

Place the coverslip containing the Fluo-4 cells in an imaging or glass-bottom chamber. Use EM buffer to wash the sample three times to remove compounds potentially resulting from cell lysis. Then, add one milliliter of EM buffer. Place the chamber on an inverted fluorescence microscope stage, heated at 33 degrees Celsius. Select a microscopy field, and set up acquisition parameters, including exposure time and binning, if necessary, to optimize the Fluo-4 fluorescent signal.

To add bacteria to the sample, carefully remove 500 microliters of EM buffer from the chamber, and add 500 microliters of the bacterial suspension to obtain a final OD₆₀₀ of 0.05. Immediately perform an acquisition, or wait 10 minutes to allow the bacteria to sediment onto the cells. At the end of the acquisition stream, acquire a phase contrast image of the selected field to visualize the bacteria contacting the cells and the membrane ruffles associated with bacterial invasion sites.

Repeat the acquisition procedure at intervals that are amenable to the duration of the image acquisitions, file savings, and selection of a new field to cover the whole process. At the end of the acquisition procedure, add to the sample, two micromolar of the calcium ionophore ionomycin to determine the maximal amplitude of the calcium signals. Acquire images every 3 to 5 seconds until the signal stabilizes, usually for less than 10 minutes.

Follow by adding the calcium chelator EGTA to a final concentration of 10 millimolar to determine the fluorescent signal in the absence of calcium. Acquire images every 3 to 5 seconds until signal stabilization, usually for less than 10 minutes. Carry out image analysis according to the text protocol.