Materials and Reagents

Materials
1. Pipette tips
2. Glass-bottomed dishes (such as MatTek, catalog number: P35G-1.5-20-C; IBIDI µ-Dish 35 mm, high Glass Bottom, catalog number: 81158; Sarstedt lumox dish 35, catalog number: 94.6077.331; Nunc Glass Bottom Dishes, catalog number: 150680)
3. 1.5-ml tubes
4. 50-ml conical centrifugation tubes
Reagents
1. HeLa cells (ATCC, catalog number: CCL-2)
2. Dulbecco’s Modified Eagle’s Medium (DMEM) – high glucose (Sigma-Aldrich, catalog number: D5796)
3. Fetal bovine serum, FBS (South America) (Biowest, catalog number: S1810)
4. Penicillin/streptomycin 100× (BioConcept-Amimed, catalog number: 4-01F00-H)
5. Trypsin-EDTA solution 10× (Sigma-Aldrich, catalog number: T4174-100ML)
6. OptiMEM-I (Gibco, catalog number: 51985026)
7. FuGENE HD (Promega, catalog number: E2311)
8. HEPES (PanReac AppliChem, catalog number: A1069)
9. CaCl₂·2H₂O (Merck, catalog number: 1.02382.0500)
10. KCl (Merck, catalog number: 1.04936.1000)
11. MgCl₂·6H₂O (Fluka, catalog number: 63064)
12. NaCl (PanReac AppliChem, catalog number: A2942)
13. DTT (PanReac AppliChem, catalog number: A1101)
14. H₂O₂ solution (Sigma-Aldrich, catalog number: 95321)
15. pCMV/myc/ER/GFP HyPerER (Enyedi et al., 2010) (Kind gift from Dr. Miklos Geiszt, Semmelweis University, Budapest, Hungary)
16. Stimulants and inhibitors (experiment-dependent), e.g., thapsigargin (EMD Millipore, catalog number: 586005)
17. HEPES Imaging Buffer (1 L) (see Recipes)

Equipment

Pipettes
Casy cell counter (Omni Life Science) or hemocytometer
Heat block for 50-ml conical tubes
Inverted microscope; we use an Olympus Fluoview3000 laser scanning microscope
60× Objective UPLSAPO60XS2 Universal Plan Super Apochromat silicone immersion objective N.A. 1.3 (N5203000)
For excitation, we use an Olympus FVL-LAS405-LX50 Laser 405 nm and FVL-LAS488-LS20 Laser 488 nm (somewhat below the excitation peaks at 420 nm and 500 nm, respectively)
Note: Monitoring of HyPer probes does not require confocality! A fluorescence microscope with suitable filter sets for excitation and emission is sufficient.
Excitation maximum of HyPer: 500 nm
Emission maximum of HyPer: 516 nm
Climate control unit; we use the Olympus CellVivo incubator system for IX83 (E0439957)
Note: Live cell experiments should be performed under optimal environmental conditions; the minimal requirement is a temperature controller to maintain the optimal temperature of 37°C; for long-term experiments, e.g., longer than 30 min, an additional CO₂ supply will be needed.

Software

FV300 (Olympus)
Excel (Microsoft)

Procedure

Cell culture and seeding
Cells should be cultured in their corresponding growth medium until they reach a confluence of around 70%.
1. Wash the cells with 10 ml 1× PBS.
2. Detach the cells with 2 ml pre-warmed (37°C) 1× trypsin (0.5 g/L) at room temperature for 4 min and resuspend in 8 ml complete growth medium.
3. Determine the number of cells in the suspension using a Casy cell counter or hemocytometer.
4. Seed 30,000 cells in 400 µl complete growth medium to the center well of a Matek glass-bottomed dish.
  Cell density may depend on the cell type and transfection method used.
5. Place the dish in a humidified cell culture incubator (37°C, 5% CO₂) and incubate overnight.
Transfection
1. Mix 25 µl Opti-MEM medium with 0.5 µg HyPerER plasmid and 1.5 µl Fugene^® HD solution (according to the manufacturer’s protocol).
2. Incubate the mixture for 15 min at room temperature and add dropwise to the cells.
3. Add 2.6 ml complete growth medium approximately 6 h after transfection.
4. Incubate the cells at 37°C for 24-48 h.
  Note: The optimal transfection conditions, e.g., cell density, DNA amount, and DNA:Fugene^® HD ratio, will require optimization depending on the cell line of choice.
Imaging
Imaging is performed on an Olympus Fluoview 3000 laser-scanning microscope with a temperature- and CO₂-controlling unit. Samples are excited sequentially using the 405 nm and 488 nm lasers. Emission is recorded in a window from 500 to 600 nm.
Note: There is only one emission window for both excitation wavelengths!
1. Take out the cell culture dish from the incubator and remove culture medium with a pipette.
2. Carefully wash the dish twice with 1 ml pre-warmed HEPES Imaging Buffer.
3. Add 1 ml HEPES Imaging Buffer and place the dish on the microscope stage.
4. Search for a field of view that contains several healthy-looking cells with sufficient YFP fluorescence signal.
5. Optimize the voltage of the photomultiplier tube to obtain good image quality (signal-to-noise) for both channels.
6. Measure the baseline oxidative status of HyPerER in the cells every 20 s for at least 5 min, then carefully add stimulating substances, such as thapsigargin (final concentration 1 µM), at double working concentration in 1 ml HEPES Imaging Buffer with a pipette and record until the signal stabilizes (or according to the stimulation protocol). Make sure to complete the addition within the 20-s time window between two image acquisitions. The speed of the acquisition and the total imaging time should be optimized to achieve proper temporal resolution but also to avoid photobleaching.
  Conclude each measurement by the addition of a single dose of saturating H₂O₂ (100 µM final concentration in 1 ml HEPES Imaging Buffer) as a positive control, carefully applied with a pipette. An example in HeLa cells is shown in Figure 1.

Data analysis

Analysis using the FV3000 software:

Define the regions of interest (ROI) in your sample and select a suitable area to place a background ROI.
Measure the raw emission intensity within these ROIs.
Export the raw intensity values from the software as .csv files.
Open the .csv files in Microsoft Excel. You will obtain two emission intensity values, determined at 516 nm, for each time point measured for every ROI, one for excitation at 488 nm and one for excitation at 405 nm. Perform the following calculation:
Subtract the intensity values of the background ROI obtained at 405-nm excitation from the target ROI measured at 405 nm.
Subtract the intensity values of the background ROI obtained at 488-nm excitation from the target ROI measured at 488 nm.
Calculate the fluorescence ratio using the following formula (for an example, see Figure 1):

Figure 1. Real-time monitoring of ER redox changes. HeLa cells were transfected with the HyPerER sensor using Fugene^® HD. At 48 h post-transfection, the cells were prepared for imaging. Fluorescence ratio changes were monitored over time. A. Each trace corresponds to the data recorded from one cell. The cells were treated with 1 µM thapsigargin (TG), which reduces the ER environment by facilitating the influx of reduced glutathione (Lizak et al., 2020), followed by the application of 100 µM H₂O₂, which leads to re-oxidation. B. Exemplary fluorescence pictures in the 405-nm or 488-nm channel and the corresponding ratiometric images. Scale bar = 10 µm. The pseudocolored images represent the ratio R, obtained by dividing the values measured at 488-nm excitation and 516-nm emission after background subtraction by the values measured at 405-nm excitation and 516-nm emission. A darker color indicates a lower ratio and hence less H₂O₂; a lighter color represents a higher ratio and hence more H₂O₂.

Recipes

HEPES Imaging Buffer (1 L)
1 M HEPES solution (sterile-filtered) 20 ml; final concentration 20 mM
1 M KCl solution (sterile-filtered) 5 ml; final concentration 5 mM
1 M CaCl₂ solution (sterile-filtered) 1.8 ml; final concentration 1.8 mM
1 M MgCl₂ solution (sterile-filtered) 1 ml; final concentration 1 mM
1 M NaCl solution (sterile-filtered) 130 ml; final concentration 130 mM
Fill to 1 L with ddH₂O and adjust pH to 7.4

Acknowledgments

This protocol was adapted from Lizak et al. (2020). Funding by the Swiss National Science Foundation (SNSF, 31003A-179400) and the University of Basel is gratefully acknowledged.

Competing interests

The authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

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