Live Mitochondrial or Cytosolic Calcium Imaging Using Genetically-encoded Cameleon Indicator in Mammalian Cells

Materials and Reagents

1. Materials
   1. Pipette tips
   2. Glass coverslip, 18-mm diameter, 0.16 mm thickness (VWR, catalog number: VWRI631-1580)
   3. 50-ml tubes
   4. 1.5-ml tubes
   5. 12-well plate
      
      
      
2. Reagents
   
   1. Dulbecco’s Modified Eagle’s Medium (DMEM)-high glucose (Merck, catalog number: D5671, store at 4 °C)
   2. Fetal Bovine Serum, FBS (Sigma-Aldrich, catalog number: F7254, store at -20 °C)
   3. Bovine serum albumin, BSA (Sigma-Aldrich, catalog number: A7906, store at 4 °C)
   4. L-glutamine (Sigma-Aldrich, catalog number: G8540, store at -20 °C)
   5. Penicillin/streptomycin 100x (Euroclone, catalog number: ECB3001, store at -20 °C)
   6. TransIT^®-LT1 transfection reagent (Mirus Bio LCC., TransIT^®, catalog number: mir2305, store at 4 °C)
   7. Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid, EGTA (Sigma-Aldrich, catalog number: E4378, store at room temperature, RT)
   8. CaCl₂ solution, 1 M (Sigma-Aldrich, catalog number: 21115, store at RT)
   9. NaCl (Sigma-Aldrich, catalog number: S1679, store at RT)
   10. KCl (Sigma-Aldrich, catalog number: P9333, store at RT)
   11. MgCl₂ solution, 1 M (Sigma-Aldrich, catalog number: 63069, store at RT)
   12. KH₂PO₄ (Sigma-Aldrich, catalog number: P5655, store at RT)
   13. HEPES (Sigma-Aldrich, catalog number: H3375, store at RT)
   14. Glucose (Sigma-Aldrich, catalog number: G7528, store at RT)
   15. Trizma Base (Sigma-Aldrich, catalog number: T6066, store at RT)
   16. Histamine dihydrochloride (Sigma-Aldrich, catalog number: H7250, powder: store at 4 °C)
   17. Thapsigargin (Sigma-Aldrich, catalog number: T9033, store at -20 °C)
   18. Cyclopiazonic acid from Penicillium cyclopium, CPA (Sigma-Aldrich, catalog number: C1530, store at -20 °C)
   19. Ionomycin calcium salt from Streptomyces conglobatus (Sigma-Aldrich, catalog number: I0634, store at 4 °C, DMSO-dissolved powder: store at -20 °C)
   20. Digitonin (Sigma-Aldrich, catalog number: D5628, store at RT; DMSO stock: store at -20 °C)
   21. Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone, FCCP (Sigma-Aldrich, catalog number: C2920, store at 4 °C; DMSO stock: store at -20 °C)
   22. Caffeine (Sigma-Aldrich, catalog number: C0750; store at RT)
   23. Succinic acid (Sigma-Aldrich, catalog number: S7501, store at RT)
   24. Plasmids:
       a.pcDNA-D3cpv (addgene, catalog number: 36323)
       b.pcDNA-4mtD3cpv (addgene, catalog number: 36324)
       c.pcDNA-D3mCeruelan3+16
       d.pcDNA3-D3mCeruelan3+16
   25. Extracellular medium (see Recipes)
   26. Intracellular-like medium (see Recipes)
   27. Intracellular-like medium for calibration (see Recipes)
   28. Intracellular-like medium for mitochondrial calibration (see Recipes)
   29. K⁺-based medium (see Recipes)
   30. 0.5 M EGTA stock solution, pH 7.4 (see Recipes)
   31. Histamine dihydrochloride (see Recipes)
   32. Thapsigargin (see Recipes)
   33. CPA (see Recipes)
   34. Ionomycin calcium salt (see Recipes)
   35. Digitonin (see Recipes)
   36. FCCP (see Recipes)

Equipment

1. Pipettes
2. Glass bottles
3. For solution preparation:
   a.Stirrers
   b.Bath
4. For FRET imaging (Figure 1)
   Fluorescence microscope with motorized dichroic turret (Leica inverted microscope DMI 6000 CS)
   Dual-pass dichroic mirror T455lp (Chroma Technology Corporation, T455lp)
   Beam-splitter, composed by:
   a.Emission filters HQ 480/40 M (for ECFP, Chroma Technology Corporation, ET480/40)
   b.Emission filters HQ 535/30 M (for YFP variants, Chroma Technology Corporation, ET535/30)
   c.Dichroic mirror 515 DCXR, Chroma Technologies (Chroma Technology Corporation, 515DCXR)
   Note: The emission filter set recommended in this protocol ensures the minimal crosstalk (or bleed-through) between the donor and acceptor emissions. An excessive crosstalk strongly diminishes the DR. Generally, the crosstalk can be estimated exploiting cells expressing only the donor and only the acceptor FP and imaging them using the filter set chosen for the FRET experiments. Of note, 30-40% of the donor fluorescence passes into the acceptor channel and this contribution is unavoidable because of the partial overlap of emission spectra of the two FPs. To remove this crosstalk mathematically, it is possible to apply spectral un-mixing (see Gordon et al., 1998).
5. Objective: 40x oil objective, HCX Plan Apo, NA 1.25 (Leica Microsystems)
6. LED 430 nm (Thorlabs, catalog number: M430L4)
   Note: LEDs are the preferred light source because of their low cytotoxicity.
7. CFP excitation, a 425/20 filter (Chroma Technology Corporation, 425/20 nm) is recommended to avoid donor and acceptor cross-excitation
8. Camera: high sensitive CCD or sCMOS camera (IM 1.4C cool camera Jenoptik Optical Systems attached to a 12-bit frame grabber)
9. Perfusion and temperature system
   Note: Ca²⁺ imaging experiments should be performed at the physiological temperature of 37 °C. To decrease the temperature differences between the chamber and the environment that can induce focal drift, it is advisable to use a combination of a perfusion system with a temperature controller, a chamber thermal stage, an objective heater and a block heater for solutions. The achievement of the correct temperature must be verified using a thermocouple probe positioned in the open-topped chamber, immersed in the experimental solution.
   1. Peristaltic pump (MINIPULS Evolution^® Peristaltic Pump from Gilson, Inc., F110701)
   2. Chamber thermal stage, (Dual Channel Temperature Controller, Warner Instruments, catalog number: TC-344C; Stage Adapters, Warner Instruments, catalog number: SA-20PLIXR-AL; Platform Interface Cables, Thermistors, and Replacement Parts; Warner Instruments, catalog number: CC-28)
   3. Block heater (Major Science; Genius Dry Bath Incubator, MD series, MD-01N/MD-02N)
   4. In-line solution heater (In-line solution heater, Warner Instruments, catalog number: SF-28)
   5. Objective heater (custom-made)
   6. Plastic tubes
      Note: Please note that some hydrophobic drugs, such as ionomycin, stick to the plastic tubes. To remove them, profuse a water-solution containing 4% BSA. However, manual addition is always the best choice for these drugs. Some cautions must be taken into account for manual addition in order to achieve a homogeneous and rapid stimulation of cells:
      1. It should be performed carefully, to avoid changing in the focus.
      2. It should be performed quickly adding a volume equal to the 10% of the total chamber volume.
   7. Thermocouple probe (Hanna Instruments, Thermometer K-type Mem-Hold Catalogue number: HI93531)
   Note: A typical perfusion system is composed by a two-way a peristaltic pump. This peristaltic pump allows the use of two tubes. The in-flow tube, connect to an in-line solution heater (controlled by the dual channel temperature controller) places immediately before the butterfly needle that allow the perfusion of solution into the chamber. The out-flow tube is instead the one devoted to remove solution from the chamber. The butterfly needles permit the employment of “play dough”, or similar, to attach the tubes to the chamber thermal stage. The speed and the resistance exerted on the tubes should be adjusted to guarantee a 2-3 ml/min of flow and to prevent the cells from drying. To maintain the temperature and avoid focal drift, a thermal stage, connected to the dual channel temperature controller, is used to adapt the open-top chamber. Finally, an objective heater can be used to avoid focal drift, if necessary. The thermocouple probe should be used to set the temperatures of the chamber thermal stage and of the in-line solution heater in order to reach 37 °C within the chamber’s solution.
10. For cell culture
    1. Incubator
    2. Oven
    3. Open-topped chambers (Warner Instruments-(QR-41LP) Quick release chamber for 18 mm coverslips, low profile, catalog number: 64-1944)
       
    
    
    Figure 1. Microscope set-up for FRET experiments. Essential components are highlighted.
    

Software

1. A software package to interface with a microscope that supports online rationing in real time, e.g.,
   MetaFluor (Universal Imaging)
   TillVision (Till Photonics)
   lidebook (SciTech)]
   We use Roboscope, a custom-made software developed by Catalin Ciubotaru at VIMM, Padua, Italy.
2. Offline analysis: e.g.,
   ImageJ (National Institutes of Health (NIH) freeware; see http://rsb.info.nih.gov/ij/)
   Excel (Microsoft) or equivalent data-analysis program
   Origin (OriginLab Corporation)
   

Procedure

1. Cell culture and transfection
   1. Plate cells on 12-well plate with an 18-mm glass coverslip on the bottom. Optimize plate density to reach approximately 40-60% of confluence within 24 h.
   2. Transfect cells with the chosen Cameleon when they are at 40-60% of confluence. For HeLa cells, we used TransIT^®-LT1 (3 µl of reagent for each µg of cDNA used) as transfection reagent and 1 µg of cDNA (the volume required depends on the cDNA concentration).
   3. Perform experiments after 24-48 h.
      
      
      
2. Optimization of the perfusion chamber
   Drug perfusion ensures the best mixing and keeps cells in fresh and warmed solution, if perfusion is correctly set. To optimize the perfusion system, follow these tips:
   
   1. Locate the pump at the same height and close to the microscope;
   2. Keep the tubes as short as possible;
   3. Set the flow rate at 2-3 ml/min;
   4. To check the flow of the perfusion system, it is possible to add a 1-2 ml solution containing phenol red (0.3 mM in water) into the chamber, attach the in-flow and out-flow tubes and perfuse water. If the chamber solution is totally exchanged, turning from red to transparent in less than 1 min, the perfusion is correctly set.
      
      
      
3. Ca²⁺-imaging experiment: SOCE activation
   1. Remove cells from the incubator, eliminate the medium and rinse cells twice with extracellular medium containing 1 mM CaCl₂.
   2. Add 1 ml of extracellular medium containing 1 mM CaCl₂ to the open-topped chamber.
   3. To focus and check cells’ health status, use bright-field illumination. Use fluorescence illumination to choose a field of view containing at least 2-4 transfected cells. To allow equilibration of cells in the saline, perfuse them with the first solution you will use for 2 min before starting the recordings session.
      Notes: The requirements that should be satisfied by the cells to image are:
      1. Donor and acceptor fluorescence needs to be at least two-fold of that of the background;
      2. Donor and acceptor fluorescence should not be the brightest since high fluorescence (typically 7 times or more the background values) level are associated with cells expressing an excess of the probe that can interfere with the cellular healthy state.
      In our microscope, we work with 200-300 ms exposure time, binning 4, 20-50% of LED intensity. However, these parameters are strongly dependent on the experimental set-up and must be adjusted to satisfy the conditions expressed at points a and b.
   4. Collect images every 20 s for a couple of min, to record the resting [Ca²⁺]. For the experiments described in this protocol, the acquisition rate is 0.5-1 Hz (i.e., one images every 1 or 2 s); however, to avoid unnecessary photobleaching, the recording rate of this step can be slowed down to one image every 20-30 s.
   5. To trigger influx of Ca²⁺ from PM (i.e., SOCE activation), the intracellular stores must be depleted. To induce store depletion, without activation of IP₃Rs, SERCA pump blockers are usually employed. Mainly two types of blockers are used: a reversible one, i.e., cyclopiazonic acid (CPA) or an irreversible one i.e., thapsigargin (TG). CPA is used at a final concentration of 20-50 µM and TG of 100 nM. Thus, treat cells with SERCA pump inhibitors at least for 7 min in a Ca²⁺-free, EGTA (600 μM)-containing extracellular medium. This step will deplete the store, allowing STIM1 puncta formation and their translocation to the PM, where ORAI1 is located. The store depletion can be performed either offline, i.e., maintaining cells at 37 °C in the oven, or recorded at the microscope. In the latter case, treatment of cells with SERCA inhibitor will cause a slow decrease of [Ca²⁺]ER that is observed as small and slow increase of [Ca²⁺] in the cytosolic and mitochondrial compartments.
   6. To allow the entrance of Ca²⁺ through ORAI1, cells are then perfused with the same medium, with or without the SERCA inhibitor (if a reversible SERCA blocker has been used, it should be added also in this saline), and challenged with a solution containing CaCl₂ 1.5 mM (Figures 2B-2E). The Ca²⁺ re-addition will cause an increase of R in the cytosolic and mitochondrial compartments.
      Note: If store depletion is achieved by adding the SERCA pump inhibitor offline, the pre-incubation with the inhibitor should be performed at 37 °C, in the absence of CO₂.
      
      
      Figure 2. Ca²⁺ imaging with a Cameleon. A. Schematic representation of a Cameleon structure and function. Ca²⁺ binding to CaM triggers its conformational change that, in turn, induces the binding of CaM to M13 forcing the CFP (donor) and the YFP (acceptor) in closer proximity (and thus increasing the energy transfer between the two FPs). B-E. Representative SOCE activation experiment in SH-SY5Y cells expressing the cytosolic Cameleon, D3cpv. Cells were pre-incubated with thapsigargin (TG, 100 nM, the irreversible SERCA blocker) for 7 min in a Ca²⁺-free extracellular medium containing EGTA 600 μM. The experiment starts perfusing cells with Ca²⁺-free extracellular medium containing EGTA 600 μM for 1 min, followed, where indicated by the same medium without EGTA and with 1.5 mM CaCl₂ instead. B. Fluorescent microscope donor and acceptor fluorescence images taken with a 40x oil objective. Scale Bar = 10 μm. C. Plot of donor (CFP) and acceptor (cpV) fluorescence emission values (arbitrary units) kinetic, subtracted of the background, of the cell indicated by the ROI. D. Plot of R-values kinetic of the cell indicated by the ROI. E. Plot of ΔR/R₀ values kinetic of the cell indicated by the ROI.
      
      
      
4. Ca²⁺-imaging experiment: PM-potential-independent SOCE
   1. Remove cells from the incubator, eliminate the medium and rinse cells twice with extracellular medium containing 1 mM CaCl₂.
   2. Add 1 ml of extracellular medium containing 1 mM CaCl₂ to the open-topped chamber.
   3. To focus and check cells’ health status, use bright-field illumination. Use fluorescence illumination to choose a field of view containing at least 2-4 transfected cells. To allow equilibration of cells in the saline, perfuse them with the first solution you will use for 2 min before starting the recordings session (see note above, Step C3).
   4. Collect images every 1-20 s for a couple of min, to record the resting [Ca²⁺]. For the experiments described in this protocol, the acquisition rate is 0.5-1 Hz; however, to avoid unnecessary photobleaching, the recording rate of this step can be slowed down to one image every 20-30 s.
   5. Since the driving force for Ca²⁺ entry is strongly affected by differences in PM potential, altering the rate and the extent of SOCE (Penner et al., 1993), the evaluation of PM-potential-independent SOCE, requires the substitution the extracellular medium with a K⁺-based medium (where NaCl is iso-osmotically substituted by KCl; see Recipes for details). In these conditions, the PM potential collapses, nullifying differences of PM potential among cells. Clearly this latter protocol can be used in cells devoid of Voltage Gated Ca²⁺ Channels because, if they were expressed, they cause a major, SOCE independent, Ca²⁺ influx.
        To trigger influx of Ca²⁺ from PM independent from PM potential (i.e., SOCE activation), the intracellular stores must be emptied using CPA or TG, as explained before. Thus, treat cells with SERCA pump inhibitors at least for 7 min in a Ca²⁺-free, EGTA (600 μM)-containing K⁺-based medium saline. As mentioned, the store depletion can be performed either offline, i.e., maintaining cells at 37 °C in the oven, or recorded at the microscope.
   6. To allow the entrance of Ca²⁺ through ORAI1, cells are then perfused with the same medium, with or without the SERCA inhibitor (when the reversible SERCA blocker has been used, it should be added also n this second buffer), and challenged with a solution containing CaCl₂ 3 mM. Indeed, since also the driving force for Ca²⁺ decreased when a K⁺-based medium is used, the extracellular [Ca²⁺] used to visualize SOCE must be doubled in respect to the classical SOCE activation protocol (from 1.5 to 3 mM). The Ca²⁺ re-addition will cause an increase of R in the cytosolic and mitochondrial compartments.
      
      
      
5. Ca²⁺-imaging experiment: ER Ca²⁺ release via IP₃R activation
   1. Remove cells from the incubator, eliminate the medium and rinse cells twice with extracellular medium containing 1 mM CaCl₂.
   2. Add 1 ml of extracellular medium containing 1 mM CaCl₂ to the open-topped chamber.
   3. To focus and check cells’ health status, use bright-field illumination. Use fluorescence illumination to choose a field of view containing at least 2-4 transfected cells. To allow equilibration of cells in the saline, perfuse them with the first solution you will use for 2 min before starting the recordings session (see note above, Step C3).
   4. Collect images every 1-20 s for a couple of minutes time, to record the resting [Ca²⁺]. For the experiments described in this protocol, the acquisition rate is 0.5-1 Hz; however, to avoid unnecessary photobleaching, the recording rate of this step can be slowed down to one image every 20-30 s.
   5. To trigger only ER Ca²⁺ release (without SOCE contribution), cells should be first perfused for 1 minute with Ca²⁺-free, EGTA (600 μM)-containing medium.
   6. Then cells are stimulated by perfusing with medium containing an IP₃-generating agonist. These drugs activate G protein-coupled receptors (GPCR) causing hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to IP₃ and diacylglycerol (DAG). The IP₃ generated mediates the release of Ca²⁺ from IP₃Rs, decreasing [Ca²⁺] in the ER and thus causing transient [Ca²⁺] increase in cytosolic and mitochondrial compartments. Different drugs can be used to elicit IP₃Rs activation depending on cell type. For HeLa cells, we use histamine at a final concentration of 10-100 µM (Figures 3A-3F) that is perfused in the extracellular medium containing EGTA, 1 minute after the perfusion of the same medium without the agonist. The usage of 100 µM of histamine ensures the maximal stimulation of IP₃R. This drug should be freshly prepared in the extracellular medium containing EGTA 600 µM or CaCl₂ 1 mM at a final concentration of 20 mM (Figures 3G-3J).
   7. The stimulus is removed, perfusing the saline without the agonist.
      
      
      
6. Ca²⁺-imaging experiment: ER Ca²⁺ release via RyR activation
   1. Remove cells from the incubator, eliminate the medium and rinse cells twice with extracellular medium containing 1 mM CaCl₂.
   2. Add 1 ml of extracellular medium containing 1 mM CaCl₂ to the open-topped chamber.
   3. To focus and check cells’ health status, use bright-field illumination. Use fluorescence illumination to choose a field of view containing at least 2-4 transfected cells. To allow equilibration of cells in the saline, perfuse them with the first solution you will use for 2 min before starting the recordings session (see note above, Step C3).
   4. Collect images every 1-20 s for a couple of minutes time, to record the resting [Ca²⁺]. For the experiments described in this protocol, the acquisition rate is 0.5-1 Hz; however, to avoid unnecessary photobleaching, the recording rate of this step can be slowed down to one image every 20-30 s.
   5. In excitable cells, it is possible to activate RyR, substituting the IP₃-generating agonist with caffeine. This drug should be freshly prepared in the extracellular medium containing EGTA 600 µM or CaCl₂ 1 mM at a final concentration of 20 mM (Figures 3G-3J). To trigger only ER Ca²⁺ release (without SOCE contribution), cells should be first perfused for 1 minute with Ca²⁺-free, EGTA (600 μM)-containing medium. The usage of 20 mM of Caffeine in the extracellular medium containing CaCl₂ 1 mM ensures the maximal stimulation of RyR.
   6. The stimulus is removed, perfusing the saline without the agonist, once the Ca²⁺ response is observed. Typically, 1-2 min of Caffeine perfusion is sufficient.
      
      
      Figure 3. Mitochondrial Ca²⁺ imaging with mCerulean3-containing Cameleon, instead of CFP. A-F. Representative mitochondrial Ca²⁺ uptake experiment in HeLa cells expressing the mitochondrial Cameleon 4mtD3mCeruelan3+16. Cells were first perfused for 1 min in medium containing 1 mM CaCl₂ followed by a Ca²⁺-free medium containing 600 μM EGTA for the same time. The IP₃-dependent ER Ca²⁺ release was triggered by perfusing 100 μM histamine (Hist), indicated by the arrow. At the end of the experiments, probe calibration was performed to measure R_min and R_max. To achieve R_min, HeLa cells were permeabilized with digitonin (Dig, 100 μM) in an intracellular-like medium containing EGTA (600 μM). To achieve R_max, an intracellular-like medium containing 5 mM CaCl₂ was perfused. A. Fluorescent microscope cpV and mCerulean3 fluorescence images taken with a 40× oil objective. Scale Bar = 10 μm. B. Plot of donor (mCerulean3) and acceptor (cpV) fluorescence emission values (arbitrary units) kinetics of the cell indicated by the ROI, subtracted of the background. C. Plot of R-values kinetic of the cell indicated by the ROI. D. Kinetics of ΔR/R₀ values of the cell indicated by the ROI. E. Plot of R % values in the cell indicated by the ROI. F. Kinetics of calculated [Ca²⁺] values in the cell indicated by the ROI. G-J. Representative mitochondrial Ca²⁺ uptake experiment in neonatal rat cardiomyocytes expressing the mitochondrial Cameleon 4mtD3mCeruelan3+16. Cells were perfused for 1 minute in an extracellular medium containing 1 mM CaCl₂ and then with the same saline containing 20 mM of caffeine to activate RyR. G. Fluorescent microscope cpV and mCerulean3 fluorescence images taken with a 40× oil objective. Scale Bar = 10 μm. H. Kinetics of donor (mCerulean3) and acceptor (cpV) fluorescence emission values (arbitrary units) in the cell indicated by the ROI subtracted of the background. I. Kinetics of R-values in the cell indicated by the ROI. J. Kinetics of ΔR/R₀ values in the cell indicated by the ROI.
      
      
      
7. Probe calibration
   The final step of the protocol is the indicator calibration, that consists in the measurement of both the minimum R, under Ca²⁺-free conditions (R_min), and the maximum R, under conditions of saturating Ca²⁺ (R_max). These R_min and R_max values represent the minimal FRET and the maximal FRET reachable with the probe. If the K_d for Ca²⁺ of the probe is known this step allows the conversion of the R-values into [Ca²⁺]. This protocol is also useful when a comparison between GECIs with different biophysical properties (i.e., different K_d and DR) is performed. For this part of the protocol, two different approaches can be used.
   
   

Digitonin-based protocol:

1. The best one consists of cell permeabilization with digitonin in Ca²⁺-free intracellular-like medium (Figures 3A-3F). Digitonin is a steroidal detergent that permeabilizes membrane, preferentially those with high cholesterol content such as the PM. The final concentration and exposure time vary depending on the organelles investigated.
2. After permeabilization, cells are perfused with the same medium without digitonin and supplemented with EGTA (600 µM), to obtain R_min.
3. Finally, cells are perfused with a saturating CaCl₂ concentration (3-5 mM) in intracellular-like medium, to obtain R_max. Of note, for mitochondrial probe calibration, FCCP should be added in each solution of the calibration step. This drug is a mitochondrial uncoupler that, collapsing mitochondrial membrane potential, allows the equilibration of extra-mitochondrial [Ca²⁺] with that in the matrix. It is used at a final concentration of 5-10 µM. This concertation ensures a complete mitochondria depolarization.
   
   

Ionomycin-based protocol

1. The second approach consists of cell treatment with 5 µM ionomycin and 3 mM EGTA, to reach R_min and then with 10 mM of CaCl₂ intracellular-like medium, to obtain the R_max. Ionomycin is a Ca²⁺ ionophore that exchanges two protons for one Ca²⁺.
2. Generally, an accurate estimation of R_min and R_max requires 4-10 min of imaging that is the average time in which R-values are stable, meaning that an equilibrium between the [Ca²⁺] of the organelle and that of the perfused saline has been reached.
   Notes:
   1. Digitonin is a detergent used to permeabilize the PM to bring the cytoplasm or the lumen of organelles in equilibrium with the perfused medium. Since the permeabilization efficiency depends on organelles’ membrane composition and cell density on the coverslips, digitonin concentration and exposure time must be assessed for each experiment in order to avoid probe leakage from the imaged cellular compartment. Thus, load cells with a cytosolic chemical probe, e.g., Fluo-4 (see Figure 4), and transfect cells with the desired Cameleon. Since Ca²⁺ ions are smaller than chemical dyes, the correct concentration and exposure time of digitonin will be the one that causes the exit of only the chemical dye, without affecting the localization and the amount of the expressed Cameleon. For HeLa cells, we used 10 µM digitonin for 30 s for cytosolic Cameleon or 50-100 µM digitonin for 1 min for mitochondrial Cameleon;
   2. To calibrate mitochondria-targeted probes it is necessary to mimic the unique features of the mitochondrial matrix. Thus, since the pH is about 8 inside these organelles, it is recommended to use an intracellular-like medium with 20 mM Tris (in substitution of HEPES) and pH 8 at 37 °C.
   3. Ionomycin use requires some cautions:
      1. In case of manual addition, use a Ca²⁺-free extracellular medium containing EGTA, at least at the same concentration of [Ca²⁺] present in the chamber medium, to achieve R_min;
      2. When, in a medium at pH 7.4, EGTA binds Ca²⁺, it releases two protons (Martell and Sillen, 1964) that cause a pH drop that sometimes can impact on both donor and acceptor emitted fluorescence. Furthermore, since ionomycin exploits the proton gradient to carry Ca²⁺, pH changes saturate the ability of the ionophore to transport Ca²⁺. To avoid this, it is possible to counteract the pH change by adding alkalinizing agents (i.e., addition of Tris-HCl), allowing further entrance of Ca²⁺;
      3. The treatment of cells with ionomycin and EGTA causes Ca²⁺ release from the ER, thus an increase in the R can be recorded with a cytosolic/nuclear Cameleon;
      4. Being a Ca²⁺ ionophore, ionomycin is saturable (Fasolato and Pozzan, 1989). Thus, it is advisable to verify the achievement of a real R_min value comparing this value with the R_min obtained by incubating cells for 30 min in extracellular medium containing 5 µM BAPTA-AM. BAPTA-AM is a Ca²⁺-specific chelator with a faster kinetic compared to EGTA;
      5. The addition of ionomycin and high [Ca²⁺] to evaluate R_max can lead to the swelling of organelles, cellular blebbing, and eventually cell death.
      
      
      Figure 4. Digitonin concentration assessment. A-B. Representative experiment in HeLa cells loaded with FLUO-4/AM. Cells were first perfused for 1 min in medium containing 1 mM CaCl₂ and then permeabilized with digitonin (Dig, 20 μM) in an intracellular-like medium containing EGTA (600 μM) for 20 s. A. FLUO-4 fluorescence images taken with a 40× oil objective (excitation light 460 nm, LED: LZ1-10DB05; LED Engin, Filer: ET470/24) and emission recorded with a 515-nm long-pass filter and a dichroic mirror (510 DCXR). Scale Bar = 10 μm. B. Kinetics of FLUO-4 fluorescence emission values (arbitrary units) in the cell indicated by the ROI, subtracted of the background. C-D. Representative experiment in HeLa cells expressing the cytosolic Cameleon, D3cpv. Cells were first perfused for 1 min in medium containing 1 mM CaCl₂ followed by a Ca²⁺-free medium containing 600 μM EGTA for the same time. The IP₃-dependent ER Ca²⁺ release was triggered by perfusing 100 μM histamine (Hist), indicated by the arrow. At the end of the experiments, probe calibration was performed to measure R_min and R_max. To achieve R_min, HeLa cells were permeabilized with digitonin (Dig, 20 μM) in an intracellular-like medium containing EGTA (600 μM). To achieve R_max, an intracellular-like medium containing 5 mM CaCl₂ was perfused. C. Fluorescent microscope donor and acceptor fluorescence images taken with a 40x oil objective. Scale Bar = 10 μm. D. Kinetics of R-values in the cell indicated by the ROI.

Data analysis

1. The collected images can be analyzed with any software that allows the choice of Region of Interest (ROI) and the measurement of the mean intensity of each ROI. A freely available example is ImageJ. Thus, open the images (.tiff format) with ImageJ and select ROIs on the background and transfected cells. Each experiment should generate, at each time point, a FRET and a donor image (see Figures 1B, 2A and 2G). These data should be exported into a data-processing program, such as Microsoft Excel.
2. Plot the time-course fluorescence values of the selected ROIs. Sometimes, an experiment should be re-analyzed, choosing ROIs based on the observed cellular responses, considering regions of spatial heterogeneity or cell-to-cell variability.
3. Once the time-course fluorescence values are imported into Microsoft Excel or Origin, the following calculations are required:
   
   1. Background (bg) correction: subtract the background mean intensity value from each mean fluorescence value. Thus, CFP fluorescence will be calculated as “CFP intensity minus bg” and the YFP fluorescence will be calculated as “YFP intensity minus bg” (see Figures 1C, 2B and 2H).
   2. From these values, the R (see Figures 1D, 2C and 2I) can be calculated as follows:
      R = (YFP intensity - bg)/(CFP intensity-bg).
   3. To amplify difference between rest condition and Ca²⁺ peaks, calculate the ΔR/R₀ (see Figures 1E, 2D and 2J) according to the following equation:
      ΔR/R₀ = (R - R₀)/R₀,
      where R₀ is calculated as the average of the R values measured in the first 5 acquisitions and R is the R at each time point.
   4. If the calibration step has been performed, R_min and R_max can be used to convert the R data into R% values (Figure 2E), accordingly to this equation:
      R% = (R - R_min)/(R_max - R_min) x 100.
   5. To convert R values into [Ca²⁺] (Figure 2F) use the K_d and n values calculated in situ (see Table 1 in Greotti et al., 2019 and Table 4 in Palmer et al., 2006). If the used Cameleon is not calibrated in situ, quantitative analyses are not recommended.
      1. If the employed Cameleon has a single K_d, use the following equation:
         [Ca²⁺] = [K_dⁿ((R- R_min)/(R_max-R))]^1/n,
         where n is Hill’s coefficient;
         For 4mtD3mCerulean3+16 (Figure 2F), the parameters used are those published in Greotti et al., 2019, i.e., the K_d is 6.18 μM, the n is 0.68, the R_min is 1.3 and R_max is 2.7.
      2. If the employed Cameleon has a double K_d, use the SOLVER tool in Excel (Palmer and Tsien, 2006).
   6. For long experiments, evaluate the bleaching.
      Note: Long experiments that last from 15 to 30 min require the evaluation of FPs photobleaching. This analysis can be done by recording the R under the same illumination and acquiring conditions that will be used during the experiments. If photobleaching is occurring, a decrease in the R over time is observed. To correct for photobleaching, multiply the intensity of the channel by a correction factor, calculated dividing the intensity of the FP at the beginning of the experiment by the FP intensity at a given time point (Zal et al., 2004).
   7. Using Origin Software, it is possible to calculate (Figure 5):
      
      1. The area under the curve (AUC) by exploiting the plugin “integrate” (for integral measurement) taking into consideration only the part of the curve that defines the rise of [Ca²⁺], to estimate Ca²⁺ content (Figures 5A-5B);
      2. The uptake rate by applying the plugin “differentiate” for derivative calculation (Figure 5C);
      3. The efflux rate by applying the plugin “fit exponential” (Figure 5D).
         
      
      
      Figure 5. Origin Software Data Analysis. A. Plot of R- values kinetics of the cell indicated by the ROI of Figure 2C. The red rectangle indicates the part of the curve analyzed by Origin software. B. Example of Area Under the Curve (AUC) analysis. In cyan the area of the curve is highlighted where the integral has been calculated and the value of the AUC elaborated by the software. C. Example of influx rate analysis. The yellow arrows indicate the initial and the ending point considered for the derivative calculation. The derivative is plotted on the right. The yellow circle indicates the maximal derivative, i.e. the maximal rate of influx. D. Example of efflux analysis. The fitting curve is in red and the red rectangle indicates the decay value to consider as efflux rate.
      
      
   8. Perform statistical analysis (Origin Software program can be used):
      1. If two groups are in comparison, firstly check the data distribution using the normality test Shapiro-Wilk.
         1)Data with normal distribution can be analyzed using the Paired Samples t-test.
         2)Data with non-normal distribution can be analyzed using Wilcoxon-Mann-Whitney test (https://ccb-compute2.cs.uni-saarland.de/wtest/).
      2. Data comparisons among three or more groups required ANOVA-test with Bonferroni post-hoc.

Recipes

Notes:

1. Ultrapure water (resistivity of 18 MΩ·cm at 25 °C) for solution preparation.
2. The following solutions can be store at +4 °C for weeks or at -20 °C for months, in the absence of glucose.
   
   

1. Extracellular medium
   135 mM NaCl
   5 mM KCl
   1 mM MgCl₂
   0.4 mM KH₂PO₄
   1 mM MgCl₂
   20 mM HEPES
   10 mM glucose
   PH 7.4, 37 °C
2. Intracellular-like medium
   130 mM KCl
   10 mM NaCl
   1 mM MgCl₂
   2 mM succinic acid
   20 mM HEPES
   PH 7.0, 37 °C
3. Intracellular-like medium for calibration
   130 mM KCl
   10 mM NaCl
   1 mM MgCl₂
   20 mM HEPES
   PH 7.0, 37 °C
4. Intracellular-like medium for mitochondrial calibration
   130 mM KCl
   10 mM NaCl
   1 mM MgCl₂
   20 mM Tris
   PH 8.0, 37 °C
5. K⁺-based medium
   5 mM NaCl
   130 mM KCl
   2 mM MgCl₂
   3 mM CaCl₂
   10 mM HEPES
   10 mM glucose
   PH 7.4, 37 °C
6. 0.5 M EGTA stock solution, pH 7.4
   Prepare a working solution of 0.5 M in water and Tris-HCl to achieve pH 7.4
   The working solution can be store at +4 °C and it is stable for months
7. Histamine dihydrochloride
   Prepare a 100 mM stock solution in water, aliquot it and store at -20 °C
   The stock solution is useable for up to one month, with a maximum of two or three thaw/freeze cycles
8. Thapsigargin
   Prepare a 1 mM stock solution in 100% DMSO, aliquot it and store at -20 °C
   The DMSO stock solution is useable for up to one month, with a maximum of two or three thaw/freeze cycles
9. CPA
   Prepare a 20-50 mM stock solution in 100% DMSO, aliquot it and store at -20 °C
   The DMSO stock solution is useable for up to one month, with a maximum of two or three thaw/freeze cycles
10. Ionomycin calcium salt
    Prepare a 1 mM stock solution in 100% DMSO, aliquot it and store at -20 °C
    The DMSO stock solution is useable for up to one month, with a maximum of two or three thaw/freeze cycles
11. Digitonin
    Prepare a 100 mM stock solution in 100% DMSO, aliquot it and store at -20 °C
    The DMSO stock solution is useable for up to one month, with a maximum of two or three thaw/freeze cycles
12. FCCP
    Prepare a 10 mM stock solution in 100% DMSO (or ethanol), aliquot it and store at -20 °C
    The DMSO stock solution is useable for up to one month, with a maximum of two or three thaw/freeze cycles
    

Acknowledgments

This work was supported by: Fondazione Cassa di Risparmio di Padova e Rovigo (CARIPARO Foundation), Veneto Region (Rete di infrastrutture e supporto dell'innovazione biotecnologica [RISIB Project]), Consiglio Nazionale delle Ricerche (CNR) Special Project Aging, Italian Ministry of University and Research (Fondo per gli Investimenti della Ricerca di Base [FIRB Project] and Euro Bioimaging Project), Telethon Italy Grant GGP16029A and Progetti di Rilevante Interesse Nazionale (PRIN).
  This protocol was adapted from our recent article (Greotti et al., 2019).

Competing interests

The authors declare no competing financial interest.

Ethics

Follow all waste disposal regulation when disposing of waste materials.
  The employment of animals must be performed in agreement with country guidelines and laws.

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