The overall goal of this fluorescent microscopy analysis using chemical probes is to analyze local and global calcium responses elicited by host cells during infection by pathogenic Shigella. This method can help answer key questions in the field of cellular microbiology such as pathogen-induced signaling leading to cytoskeletal reorganization or cell death. The main advantage of this technique is that by using chemical probes one can follow local calcium signals associated with local processes diverted by the pathogen.
Generally individuals new to this method will struggle because of the setting of parameters that enable to follow local calcium signals while preserving the integrity of biological samples. To begin, plate wild-type Shigella expressing the AfaE adhesion onto a trypticase soy or TCS agar plate containing 0.01%Congo red and incubate the plates at 37 degrees Celsius for 18 hours. Prepare pre-cultures from the streaked plates by picking three red colonies and inoculating TCS broth with 75 micrograms per milliliter of ampicillin.
Grow the liquid cultures in a shaking incubator at 37 degrees Celsius and 200 rpm for 16 hours. Next, inoculate TCS with the bacterial pre-culture at a 1:100 dilution. Incubate the culture at 37 degrees Celsius in a shaking incubator at 200 rpm for two hours.
Ensure that the optical density at 600 nanometers is 0.2 to 0.4. Centrifuge the culture at 13, 000 g and 21 degrees Celsius for two minutes and re-suspend the pellet in an equivalent volume of EM medium. Dilute the bacterial suspension in EM buffer at a final OD 600 of 0.1 and use it immediately or store it at 21 degrees Celsius and use it within the next 60 minutes.
Grow HeLa cells in DMEM with one gram per liter of glucose supplemented with 10%FCS and grow the culture at 37 degrees Celsius with 10%CO2. Grow TC7 cells in DMEM with 4.5 grams per liter of glucose, 10%FCS, and non-essential amino acids and culture at 37 degrees Celsius and 10%CO2. For cell maintenance, split the cells regularly to avoid growth contact inhibition linked to confluent states.
This is critical for high calcium probe loading and transfection efficiency. Trypsinize and count the TC7 cells. The day before the experiment with HeLa cells, plate the cells onto sterile, 25-millimeter diameter circular cover slips in six-well plates at a density of three times 10 to the five cells per well.
Then, to allow for polarization before the experiments, seed the cells at four times 10 to the five cells per well and incubate them for five to seven days, replacing the medium every day. On the day of the experiment, remove the medium and use one milliliter of EM medium to wash the cells three times. Load the cells with three micromolar Fluo-4 AM and EM buffer and incubate the plates at 21 degrees Celsius for 30 minutes.
With EM buffer, wash the samples three times, then add one milliliter of EM buffer, and incubate the cells at 21 degrees Celsius to allow for the hydrolysis of the AM moiety. Place the coverslip containing the Fluo-4 loaded 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 OD 600 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 three to five 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 three to five seconds until signal stabilization, usually for less than 10 minutes. Carry out image analysis according to the text protocol.
In this experiment, Fluo-4 loaded HeLa cells were challenged with wild-type Shigella and stream acquisitions were performed to analyze calcium signals. A time-lapse image series of the fluorescence intensity of the Fluo-4 probe is averaged in a region of interest for a single cell, and the corresponding phase-contrast image is shown. Atypical local increases in free cytosolic calcium are observed at the Shigella invasion site with a varying amplitude and durations ranging from 2.5 to five seconds, followed by global increases in the infected cell.
In this example, an isogenic mutant strain, deficient for the type III effector IpgD, a phosphatidyl 4, 5-bisphosphate phosphatase, induced more global and less atypical local responses with long durations as compared to the wild-type Shigella strain. As seen in this experiment, the imaging of global calcium responses over extended infection kinetics shows a decrease in the frequency of responses 30 minutes following the challenge with wild-type Shigella. Finally, the quantification of dose-dependent cell responses to the calcium agonist histamine illustrates the inhibition of an inositol triphosphate dependent calcium release at late stages of a Shigella infection.
Following this procedure, all the methods simultaneously combining compatible fluids and imaging can be performed in order to address the role of calcium signals in a given process. Don't forget that working with pathogens can be extremely hazardous, and precautions such as waste disposal, cleaning of contaminated surfaces or equipments should always be taken while performing this procedure.
