Methods

3.1. Transfection 1. Seed HeLa cells onto 35-mm glass-bottom dishes in D-

MEM supplemented with 10% FCS.

2. Allow the cells to grow to 70-80% confluence.

3. Change media for Opti-MEM without serum, 700 pL per dish. Incubate 1 h. Steps 4-7 describe the protocol for one 35-mm glass-bottom dish:

4. To a sterile eppendorftube (1.5 ml) add 100 pl serum free Opti-MEM. Add 3 pl of FuGENE 6 transfection reagent (see Note 1). Mix rapidly by vortexing, and allow to stay for 5 min.

5. Add 2 pl (1 pg) of HyPer-Cyto vector. Mix rapidly by vortexing, allow to stay for 15 min for liposome-DNA complex to form.

6. Add the content of the tube to the media covering the cells in the glass bottom dish. Mix gently.

7. Add FCS to reach final concentration of 2%. Mix gently. Place the dish into a CO incubator.

3.2. Imaging of H2O2 Induction in HeLa Cells Stimulated With EGF

1. Sixteen to 24 h after transfection, check the effectiveness of transfection by using excitation with a mercury lamp. Use the GFP filter set in "visual" mode of the microscope. In fact, it is not necessary to get 100% of the cells transfected, but the more fluorescent cells that are in a field of view, the more information about the synchronicity of the cells ' response during imaging series is obtained.

2. Experiments can be carried out without a CO2 and temperature control chamber. One hour before imaging, change Opti-MEM supplemented with 2% FCS for 1 mL of L15 media supplemented with 2% FCS and allowed the dish to stay at room temperature.

3. Place the dish on the microscope table (seeNote 2), and find a group of transfected cells by using the visual mode of the microscope. Once the cells are in the field of view, immediately close the lamp shutter to avoid unwanted bleaching of the probe.

4. Switch the microscope to the scanning mode and activate the following beam path settings, or equivalent depending on the manufacture of the microscope:

a. "Mode"—xyt. This mode is used for acquisition of time series for two-dimensional images (see Note 3).

e. Scan direction—unidirectional.

f. PMT voltage—730-800. Lower value makes pictures less noisy; higher value lets the use of less laser power.

h. Excitation Beam Splitter DD 488/543.

i. Emission wavelengths—500-530 nm.

J. "Speed" (Beam frequency) 400 Hz. If available; optionally, "line average 2" function can be activated to make pictures less noisy.

Set number of frames to be acquired and delay between frames using "Time" function. For growth factors stimulation experiments usually the use 1-10 s delay is recommended. All imaging experiments usually take 1-2 h.

Zoom selected cells with "Zoom in" function.

5. Perform several single scans to set final focus; adjust laser power and PMT voltage. Press "Start Series" button. The YFP that is the fluorescent core of HyPer tends to photo-convert into the dark state upon irradiation with excitation light. After several seconds in the dark state, YFP chromo-phore turns back into the fluorescent state. At every single moment, there is a dynamic equilibrium between fluorescent and dark states that depends on excitation light power and frequency of frames acquisition. In fact, during the several frames after the start of a time series, fluorescence goes down and after the equilibrium between fluorescent and dark states of YFP is stable fluorescence no longer changes. Press "Stop Series" button and than immediately press "Start Series" button again. During this time the next 10-20 frames will give a "baseline". Acquiring a series can be then achieved by activating "Quantification-stack profile" window of Leica Confocal Software (LCS) software.

6. Dilute 1 pL of working solution of EGF in 50 pL of L-15 media and carefully, drop-by-drop add it to the dish. Spread single drops to "cover" all surface. Do not touch the dish during addition. If you use the liquid perfusion module (see Note 5), perfuse L-15 media with the same concentration of EGF. Mark the time point of addition.

7. Stop the acquisition at the appropriate time.

3.3. Quantification Quantification can be easily performed with the standard LCS

of Results software.

1. Press the "LUT" button. Select "P.color 7" lookup table (see Note 6).

2. Activate "Process" window in LSC software, select "Enhancement" window, then choose the "Baseline correction" option to allow subtract background. After background subtraction, fluorescent signal from the cells can be quantified.

3. Open "Quantify" window of LCS software (Fig. 6.1). Activate "Stack profile" window. Press "Viewer" button.

4. Select regions of interest.

5. Press "Graphs" button. In the window that appears the time profile of fluorescence intensity in selected regions of interest will be seen. Activation of the right mouse button menu on the graphs allows the exporting of graphs to ".txt" or ".jpeg" formats (see Note 7).

Fig. 6.1. Quantification of HyPer imaging series. (A) Screen shot from Leica Confocal Software "Stack Profile—Viewer" window. In this window all the stack can be viewed. The left upper buttons allow differently shaped regions of interest to be defined. By clicking the "Graphs" button above the image, time course (B) of signal intensities in selected regions of interest appears. Activating the right mouse button menu on the graphs allows the export of graphs to ".txt" or ". jpeg" formats. Pseudo-colored images of HeLa cells expressing HyPer-Cyto at the time point of (C) and 1 h after (D) EGF addition are shown (See Color Plates).

Fig. 6.1. Quantification of HyPer imaging series. (A) Screen shot from Leica Confocal Software "Stack Profile—Viewer" window. In this window all the stack can be viewed. The left upper buttons allow differently shaped regions of interest to be defined. By clicking the "Graphs" button above the image, time course (B) of signal intensities in selected regions of interest appears. Activating the right mouse button menu on the graphs allows the export of graphs to ".txt" or ". jpeg" formats. Pseudo-colored images of HeLa cells expressing HyPer-Cyto at the time point of (C) and 1 h after (D) EGF addition are shown (See Color Plates).

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