Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Summary

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca²⁺ indicator dyes in the ER lumen.

Abstract

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca²⁺ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca²⁺ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca²⁺ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca²⁺ indicator and a hydrophilic fluorescent dye/Ca²⁺ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F₀.

Introduction

In order to resolve physiological calcium responses of the ER, we developed a new strategy to improve the trapping of synthetic calcium sensitive dyes into the ER. The method enables the direct, non-disruptive real-time monitoring of free ER calcium in presence of extracellular calcium.

Function and signaling of ER Calcium

Calcium signals are found in different cell types, e.g. muscle-cells, neurons and glial cells and their functions range from mediating muscle contraction to an involvement in synaptic transmission in learning and memory ^1,2. Changes in free calcium concentration are of high scientific interest because calcium is involved in the regulation of gene transcription, cell proliferation, neuronal excitability, cell death and other cell signaling events ^1-7. All these cellular calcium signals are functionally connected and contribute to intracellular calcium store dynamics ^8-10.

A common feature among all calcium signals is the flow of calcium between the extracellular space, the cytosol and organelles, mainly the ER and mitochondria. This causes dynamic changes in calcium concentration within these organelles, which are sensed by different signaling components. In general, the calcium concentration in the ER ranges between 100 to 800 μM, in the cytosol the calcium concentration is close to 100 nM, and in the extracellular space the concentration is around 1-2 mM. Accordingly, there is a high chemical driving force for calcium flow towards the cytosol ^2,9,10.

The most commonly investigated ER-derived calcium signals depend on the stimulation of G-protein coupled receptors (GpcR), which then activate phospholipase C (PLC). PLC in turn produces inositol 1,4,5-trisphosphate (IP₃) ¹. Upon binding of IP₃ to its receptor (IP₃-Rec, Figure 1) in the ER-membrane, calcium ions are released from the ER lumen. Historically, IP₃-mediated calcium release from the ER was first - even though indirectly - measured in acinar pancreatic cells by Streb et al. in 1983 ¹¹. This publication suggested for the first time a signaling cascade involving acetylcholine, phospholipase C, and IP₃. This way of calcium release is generally termed IP₃-induced calcium release (IiCR) (Figure 1). Kinase-dependent activation of phospholipase Cγ by receptor tyrosin kinases links the action of growth factors and neurotrophic factors to ER calcium signaling after IP₃ elevation ¹². In addition to IiCR, calcium elevation may be mediated by ionotropic calcium entry, for instance via voltage-gated calcium channels (Ca_V), and subsequent calcium-induced calcium release (CiCR) by ryanodine receptors (RyR). IiCR and CiCR are physiologically linked to store-operated calcium entry (SOCE). SOCE includes the action of STIM (stroma interacting molecule), which is a sensor for ER calcium release. STIM has been shown to stimulate extracellular calcium entry through transient receptor potential channels (Trp) ¹³, Orai calcium channels ¹⁴ and even voltage-gated calcium channels ¹⁵ (Figure 1) . Loss of ER calcium is dynamically rescued by the action of the sarco-endoplasmic reticulum calcium ATPase (SERCA), which actively pumps calcium back into the ER. Blocking the SERCA with drugs such as thapsigargin unveils a continuous loss of ER calcium to the cytosolic compartment. This ER calcium "leak" is caused by ER intramembrane pore complexes such as the Sec61 protein complex ^16,17 (Figure 1).

In 1998, Berridge published a model, the "neuron within a neuron model", which suggests a principle physiological role of the ER in integrating neuronal calcium ⁵. This model considers the existence of a continuous ER membrane system forming an intracellular "image" of the neuronal plasma membrane ⁵. This binary eukaryotic membrane system was claimed to be a basic prerequisite for temporal and spatial integration of fast and slow calcium signals in neurons. Calcium signals that occur either concomitantly or subsequently in different spines or dendrites of the same neuron are conferred to the cell's soma or nucleus via the ER, where they are summed up ^5,18. Then, their sum may have effects on the excitability of the neuron, regulation of gene transcription or integration of signaling cascades. Thus, the ER supports the integration of calcium signals. One prerequisite for this concept is the continuity of the ER in a single cell, which has been claimed by several studies and which has been proven at least for somato-dendritic areas and short distance axonal projections ^19-21. Whether there is ER continuity within long axonal projections is a matter of debate.

Strategies to measure the flow of free calcium over the ER membrane

Calcium signals are most frequently monitored in the cytosol ^22,23. Therefore, it cannot easily be distinguished whether Ca²⁺ is flowing into the cytosol from extracellular or from intracellular stores ^6,24. To overcome this limitation, methodological strategies for direct ER calcium imaging have been developed. In summary, the following strategies are used: (1) ER-targeted genetically engineered protein-indicators ^25-27. Protein-based low-affinity Ca²⁺ indicators use the bioluminescent protein aequorin or GFP in combination with a calcium sensing protein. These genetically engineered Ca²⁺ indicators (GECIs) can be targeted to the ER with the help of a signal peptide and are actively kept in the ER using a retention and retrieval motif. Common ER Ca²⁺ indicators base on the Cameleon principle and are the Cameleon YC4.3 ^26,28; Cameleon split YC7.3ER ²⁹, and Cameleon D1 ³⁰. (2) Direct esterase-based dye loading of AM-ester low-affinity Ca²⁺ indicators ^31,32. AM-derivatives of the indicator dyes (Mag-Fura2-AM, Mag-Fluo4-AM or Fluo5N-AM) pass the biological membranes in a lipophilic, calcium-insensitive state. Then, in the cytosol as well as in the ER, endogenous esterases cleave of the AM-ester group and release the Ca²⁺ indicator, leaving behind a certain amount of active dye in the cytosol and in the ER. Therefore, this approach is useful under conditions of a high calcium concentration in the ER as long as the cytosolic calcium concentration stays well below the detection limit of the low-affinity indicators, particularly during characteristic calcium signals (e.g. nM to low μM). (3) AM-ester loading in combination with plasma membrane permeabilization ³². Any remaining cytosolic Ca²⁺ indicator is removed by plasma membrane permeabilization with small amounts of a "mild" detergent (e.g. saponin) in an artificial intracellular buffer. Thus, the intracellular membranes may be stimulated, e.g. with IP₃ in the intracellular buffer, directly through "pores" in the plasma membrane. (4) Dialysis of the cytosol under whole-cell configuration and simultaneous measurements of Ca²⁺ in the ER lumen and the cytosol ^32,33. A cell is first loaded with a low-affinity Ca²⁺ indicator (e.g. Mag-Fura2-AM, ratiometric, UV-light). Afterwards, with the help of a patch pipette, any remaining cytosolic low-affinity Ca²⁺ indicator is dialysed out of the cytosol with a buffer containing a high-affinity Ca²⁺ indicator (e.g. Fluo-3, visible light). This strategy enables the simultaneous recording of cytosolic and ER derived signals. (5) Targeted-esterase-induced dye loading ^8,34. A Carboxylesterase (CES) is targeted to the lumen of the ER and provides a high esterase activity for efficient trapping of the AM-ester form of low-affinity Ca²⁺ indicators.

Targeted-esterase induced dye loading (TED)

To improve targeting of low-affinity Ca²⁺ indicators to the ER lumen, TED was developed. TED requires the overexpression of an ER targeted mouse carboxylesterase (CES2) (Figure 2), which is achieved via expression constructs. Cells expressing a recombinant CES-construct are incubated with the AM-ester form of a calcium indicator dye (Fluo5N-AM, Figure 2). Then, in the ER, the dye is converted to the Ca²⁺ sensitive, membrane impermeable Ca²⁺ indicator complex (Fluo5N/Ca²⁺) by the high esterase activity, therefore trapping the dye in a high concentration in the ER lumen ^8,34. The method is especially useful to investigate ER calcium-release via the IiCR-pathways for instance via metabotropic, purinergic- or glutamate receptors ^8,34 and to visualize ER calcium depletion via "leak channels" directly, for instance after blockade of the SERCA ^17,34. To our experience the low-affinity Ca²⁺ indicator Fluo5N-AM is currently the best available indicator to use with the TED dye loading strategy. Fluo5N-AM has a low-affinity for Ca²⁺ (dissociation constant K_D ~90 μM, Figure 2), is almost non-fluorescent in its AM-form, but provides high fluorescence emission upon calcium binding ^8,34. The Fluo5N/Ca²⁺ complex can be excited with a standard light source of ~490 nm, which corresponds to standard dyes such as FITC, Alexa 488, or eGFP. Cytosolic calcium signals scarcely reach a concentration in the low μM range and are therefore barely detected by Fluo5N in the cytosol ³⁵.

To improve the TED performance, several recombinant vector constructs were developed (Figure 3). Originally, TED vectors based on the coding sequence of CES2 (Refseq accession number NM_145603, CES2c) and best TED performance is observed with stable expression of CES constructs. New TED vectors express a core element of CES2 fused to the red fluorescence protein TagRFP-T2 ³⁶. These vectors have the advantage that they can be used to identify transduced cells and to use the red fluorescence as an internal control for the normalization of changes in Fluo5N/Ca²⁺ fluorescence. The red fluorescence also offers the possibility to visualize the structural distribution of the ER and changes in ER dynamics under stimulation conditions.

Protocol

This protocol introduces TED applied to cell lines, hippocampal neurons and cortical glial cells. TED performance is best when TED vectors are stably expressed, for instance by lentiviral vectors. A schematic overview of the TED method is shown in Figure 4.

1. Preparation of Solutions

The following solutions should be prepared before starting and can be stored as indicated. We generally use sterilized glass and plastic material and autoclaved water.

1. Prepare HEPES-Ringer (in mM): 125 NaCl, 3 KCl, 25 HEPES, 2 MgSO₄.7H₂O, 2 CaCl₂, 1.25 NaH₂PO₄.H₂O, 10 glucose.H₂O. Calcium-free HEPES-Ringer consists of (in mM): 127 NaCl, 3 KCl, 25 HEPES, 2 MgSO₄.7H₂O, 1.25 NaH₂PO₄.H₂O, 10 glucose.H₂O and 0.1 EGTA. Without glucose these imaging solutions can be stored at 4 °C. The required amount of D-glucose is freshly added before use.
2. For TED analysis in hippocampal neurons, artificial cerebrospinal fluid (ACSF) is recommended. ACSF is composed of (in mM): 127 NaCl, 23 NaHCO₃, 3 KCl, 2.5 NaHPO₄.H₂O, 25 D-glucose.H₂O, 2 CaCl₂, 2 MgCl₂.7 H₂0 in ddH₂0.
3. Prepare Fluo5N-AM to a concentration of 5 mM. To help solubilization add 8.9 μl of 20% Pluronic F-127 (in DMSO, stored at room temperature, protected from moisture and light) to 50 μg of lyophilized Fluo5N-AM. Then solubilize Fluo5N-AM by means of a water-bath sonicator for at least 2 min. Store aliquots à 0.5 μl at -20 °C, protected from light and moisture.
4. Prepare antagonist and agonist stocks as needed. For instance: a) 10 mM ATP or ADP (in H₂O, metabotropic activation of purinergic receptors), b) 10 mM carbachol in H₂O (agonist of muscarinic acetylcholine receptor), c) 30 mM cyclopiazonic acid (CPA) in DMSO (blocker of the SERCA), d) 50mM DHPG in PBS (metabotropic glutamate receptor agonist for mGluR_I and mGluR₅), e) 10 mM Glutamate in H₂O, f) 1 mM ionomycin in DMSO (ionophore), g) 5 mM thapsigargin in DMSO (high-affinity blocker of the SERCA). Store aliquots at -20 °C.
5. Lentiviral vectors: This protocol includes the use of lentiviral TED expression vector particles. Their production and storage is not part of this protocol. We use lentiviral vectors of the second generation for the transfer of recombinant Carboxylesterases to cell lines, glial cells and neurons. Our lentiviral system bases on the expression vector FUGW ³⁷, and the packaging plasmids pCMVΔR8.91 or psPAX2 and the pseudotyping plasmid pMD2.G ^38,39. CAUTION: Consider that the work with recombinant self-inactivating lentiviral vectors requires the careful consideration of biosafety guidelines. In many countries, these vectors are classed as biohazard risk group 2. For more information refer to http://www.addgene.org/lentiviral/.

2. Preparation and Viral Infection of Mouse Hippocampal Neurons

1. Wash glass coverslips in a glass Petri dish of 20 cm diameter (100 coverslips per dish) 1x in 70% ethanol for approximately 30 min. Finally, add 100% ethanol (p.a.) for 2 min. Remove residual ethanol and dry the coverslips under a sterile hood.
2. Prepare two different kinds of media for hippocampal neuron cultures: (a) neurobasal medium with B27 1:50; (b) full medium: neurobasal medium with B27 1:50, Glutamax 1:100, and N2 supplement 1:100. Sterile filtrate the media and store them in the cell incubator until use.
3. One day prior to dissection, the coverslips are prepared by first placing one sterile 10 mm coverslip in each well of a 4-well cell culture dish. Afterwards, carefully coat each coverslip with 80-100 μl 0.1% Poly-L-lysine and store the dishes in an incubator over night.
4. At the day of dissection and cell culture, begin with washing the coverslips three times with 100 μl HBSS and transfer 100 μl neurobasal medium to each coverslip. Place the dishes into an incubator for equilibration (e.g. during dissection of mice).

Dissection

We perform our experiments with mice in accordance with European Union guidelines, as approved by our institutional animal care and utilization committee.

5. Remove the total brain and dissect the hippocampi bilaterally.
6. Carefully remove any meninges and tissue other than the hippocampus and place one hippocampus each in a 1.5 ml tube containing 450 μl HBSS. Store the tissue on ice until a sufficient number of hippocampi has been isolated.

Cell culture

7. Add 50 μl 1% trypsin (Worthington) to each tube and incubate them in a 37 °C water bath for 15 min. Shake the tubes occasionally. To stop the digestion reaction, add 50 μl 1% trypsin inhibitor to each tube and invert the tube several times.
8. Transfer all tissue of up to 5 hippocampi to one 15 ml falcon tube avoiding the transfer of liquid. Add B27-medium (a) to a final volume of 5 ml.
9. Triturate the tissue using a fire polished Pasteur glass pipette by carefully pipetting up and down. CAUTION: Avoid air bubbles!
10. Spin with 400 x g for 3 min, aspirate supernatant and resuspend the cell pellet in 5 ml B27-medium (a).
11. Triturate the tissue once again with the glass pipette, and then twice with the help of a 1,000 μl plastic filter tip. Perform this as described in steps 2.9 and 2.10.
12. After the last centrifugation, resuspend the cell pellet in 2 ml full medium (b) and triturate cells with a 200 μl plastic filter tip. To separate remaining cell clumps from the single-cell suspension, let cell clumps settle down for 1-2 min.
13. Count cells and calculate the total number of cells needed for viral infection. For TED imaging use 25,000 cells per 10 mm coverslip.
14. Plating additional 25,000 non-transduced cells as a control is recommended at this point.

Viral Infection

15. Transfer the cell suspension for viral infection into a fresh 15 ml falcon tube and centrifuge for 3 min at 400 x g.
16. Aspirate the supernatant and resuspend the cells in 300 μl medium (b)
17. Add an appropriate amount of infectious lentiviral TED expression vector particles and let the falcon tube stand at room temperature for 10 min.
18. Fill up the tube with full medium (b) to the final volume needed for plating (100 μl for each 10 mm coverslip), aspirate the medium from the coverslip, and immediately place 100 μl cell suspension onto each coverslip.
19. Place the dishes into the incubator until all cells have settled down (approx. 2 hr) and carefully fill up the dishes until they contain 2 ml medium (b).
20. Let neurons grow for at least one week. Replace 50% of the medium every week.

3. Culture and Viral-infection of Mouse Cortical Glial Cells

Preparation

1. Start by preparing basal glia medium (1:1 mixture of DMEM/F12 with 10% FCS, 5% HS, 1% penicillin/streptomycin, 0.45% glucose) and coating of a T75 cell culture flask with 4 ml 0.5 ng/ml Poly-DL-ornithine hydrobromide (PORN) (diluted in 150 mM boric acid solution, pH 8.35). After incubation at 37 °C for 2 hr or over night, wash the cell culture flask three times with HBSS and add 18 ml basal glia medium containing B27 1:50 and 10 ng/ml EGF. Equilibrate the cell culture flask in the incubator (up to 3 hr).

Dissection

1. We perform our experiments with mice in accordance with European Union guidelines, as approved by our institutional animal care and utilization committee.
2. Dissect the brain of one P5-P7 mouse and remove the hippocampus and meninges from both hemispheres as described in 2.5.
3. Dissect the frontal cortex, cut it into several small pieces and collect them in a 1.5 ml tube containing HBSS. Store the tube on ice and proceed to the cell culture part.

Cell culture

4. Transfer all tissue from the tube to a 15 ml falcon tube using a fire polished glass pipette.
5. Add basal medium to a final volume of 5 ml and triturate the tissue by pipetting several times up and down with a fire polished glass pipette. After centrifugation at 300 x g for 3 min, aspirate the medium, and resuspend the cell pellet in 5 ml basal medium.
6. Repeat step 3.5 twice.
7. After the third centrifugation, resuspend the cell pellet in 2 ml basal glia medium containing B27 (1:50) and 10 ng/ml EGF.
8. Titruate once again and transfer the cell suspension to the prepared T75 cell culture flask and let cells grow for 3-4 days.

Washing of glial cells (after 3-4 days in culture):

9. Wash cells once with 10 ml PBS and vigorously tap or gently hit the flask a few times with your hand, while holding it tight in the other hand. By this step cell debris and cell clusters are removed from the culture.
10. Aspirate the PBS and add fresh basal glia medium containing B27 (1:50) and 10 ng/ml EGF. Further cultivate the culture for 5-7 days until the cells reach 80% confluency.

Splitting, transduction and final seeding of glial cells:

11. Coat 10 mm coverslips with 100 μl Poly-D-lysine (Stock solution: 0.1%, diluted 1/50 ad 20 μg/ml in PBS or HBSS) and incubate them in an incubator over night. Wash coverslips three times with HBSS and add 100 μl basal glia medium. Equilibrate coverslips in the incubator (3 hr).
12. Wash the glial cells twice with 10 ml PBS, aspirate the PBS, and add 3 ml trypsin (TrypLE, undiluted). Trypsinize cells for up to 1 min. CAUTION: Avoid over-trypsinization. Normally more than 80% of all cells are detached after 1 min and this is sufficient to have several million of healthy cells.
13. Stop the reaction by adding 10 ml basal glia medium, transfer the cell suspension to a 15 ml falcon tube, centrifuge at 300 x g for 3 min. Aspirate the supernatant and resuspend the cell pellet in 5 ml basal glia medium.
14. Again, spin and aspirate as described in step 3.15, then resuspend cells in 5 ml basal Glia medium.
15. Transfer the required number of cells to a 1.5 ml tube. 10⁴-2x10⁴ glial cells are sufficient for one 10 mm coverslip. Usually, the suspension volume to transduce cells for one 4-well-dish is less than 200 μl. Add an appropriate amount of lentiviral TED expression vector particles to the cells and incubate them at RT for 10 min. Then fill up the suspension with basal glia medium to the seeding volume (100 μl per coverslip).
16. Seed 100 μl of infected cells per coverslip and incubate for roughly 2 hr, then add basal glia medium containing B27 1:50 and 10 ng/ml EGF to a final volume of 2 ml.
17. For ideal culture conditions use cells for experiments on day 3-7 after plating.

4. Generation and Culture of Reporter Cell Line

TED reporter cell lines were established on the basis of HeLa, BHK21, Hek293 and SH-SY5Y. Here we provide the protocol for HeLa cells, but the protocol may be transferred to any other cell line as well.

Generation of stable HeLa cell line

1. Split a wild type HeLa cell line and transfer 100,000 cells in a volume of 200 μl to a 1.5 ml tube.
2. Add an appropriate amount of lentiviral TED expression vector particles to the tube and incubate the cell-virus suspension for 10 min at RT. Then seed the cells in a total volume of 2 ml medium (DMEM, 10% FCS, 1% penicillin/streptomycin) in a 30 mm dish and incubate for 3 days.
3. After washing once with PBS, aspirate medium and add 500 μl trypsin (TrypLE, 2:3 in PBS). Incubate for approx. 3 min and then stop the reaction by adding 3 ml medium. Transfer the suspension into a 15 ml falcon tube and centrifuge at 400 x g for 3 min.
4. Resuspend the cell pellet in 200 μl medium and again infect the cells with lentiviral particles as described in 4.2. Then, seed cells in a T25 cell culture flask in 10 ml medium.
5. Grow cells until a confluency of 60-80% is reached. Then split the culture.

TED reporter cell lines

6. TED reporter cell lines can be maintained in any standard medium, e.g. DMEM containing 5% or 10% FCS and 1% penicillin/streptomycin is used.
7. Plate an appropriate number of cells in a 4-well dish containing sterile 10 mm coverslips (see above), e.g. 10,000 to 20,000 cells per coverslip.
8. Grow cells for at least two days before using them for TED imaging.

5. Preparation for the Live Imaging Procedure at an Inverted Microscope

1. 1. Prewarm HEPES-Ringer to room temperature and add D-glucose.
   2. Prewarm the ACSF (without Ca²⁺ and Mg²⁺⁾ to room temperature and add D-glucose. Aerate the ACSF with carbogen for 5 minutes. Then add 1 M stock solutions of CaCl₂ and MgCl₂ to final concentrations of 2 mM each.
2. Start the live imaging microscopes and all computer programs.
3. Start perfusion on a "dummy" imaging chamber and equilibrate the tube system.
4. Prewarm the inline solution heater and the imaging chamber in the microscope stage. Fix the imaging chamber in a heating insert.
5. Equilibrate the system to 32-37 °C before you start the dye loading procedure. CAUTION: To avoid accidental flow of perfusion solution into the microscope system we use hair ties around the objectives and elastomeric silicone sheets (1mm) to protect the microscope stage.

6. Dye Loading of Either Cell Type

1. Resuspend 1 aliquot of Fluo5N-AM (see 1.3) in 100 μl prewarmed imaging solution (HEPES-Ringer or ACSF).
2. Solubilize Fluo5N-AM in the imaging solution (HEPES-Ringer or ACSF) in a water-bath sonicator for 90 sec.
3. Use a fresh 4- or 24 -well plate and transfer 400 μl prewarmed imaging solution to one well. Add the dye solution to the same well to come to 500 μl of a 5 μM solution.
4. Carefully place a coverslip with cells (e.g. 10-18 mm in diameter) in the well, cells facing up.
5. Incubate for an appropriate period of time in a cell culture incubator at 37 °C (meanwhile prepare imaging settings at the microscope stage). Typical dye-loading times are for neurons and glial cells 7-15 min and for cell lines 10-20 min.

CRUCIAL: Dye-loading time and dye concentration depend on the cell type, cell density, and experiment-specific needs! Long incubation times in presence of the dye may harm the cells, especially primary neurons and primary glial cells and this is critical for the responsiveness to physiological stimuli. Long-incubation times (e.g. 30 min) will only be useful for ER calcium localization experiments to investigate the distribution of ER calcium in small subcellular structures, e.g. fine neurites or spines. In some experiments, a mixture of 1/3 growth medium with imaging solution may help to protect cells during Fluo5N-AM loading.

6. Instantly, transfer the coverslip to another cell culture well containing imaging solution (HEPES-Ringer or ACSF) and start mounting the cells into the imaging chamber.

7. ER-calcium Live Cell Imaging with an Inverted Laser Scanning Confocal Microscope

1. Use a paint brush to apply high vacuum grease to a imaging chamber. The grease is needed to fixate the coverslip onto the bottom of the perfusion chamber. We use self-made imaging chambers for 10-12 mm coverslips with a small volume, thus allowing high perfusion speeds of up to 15-fold buffer exchange per minute. A commercially available perfusion chamber for 18 mm coverslips, may be purchased from Warner instruments (RC-49FS). This chamber also enables field stimulation of neurons.
2. Pick up the coverslip with the Fluo5N-loaded cells and add a small drop of imaging solution (50-100 μl) onto the cells to keep cells covered with imaging solution. Turn the coverslip upside down and mount the cells into the imaging chamber. To press the coverslip into the silicon-glue, use a cotton bud (e.g. Q-tips).
3. Wipe away residual buffer from the bottom of the glass coverslip with cotton buds. CAUTION: Carefully wipe away residual moisture when using an oil-objective with high numerical aperture for high-resolution imaging. Residual salt is best removed by water. Accidental smear of grease may be removed with acetone or pure ethanol.
4. Exchange the "dummy" imaging chamber with the experimental imaging chamber. Wash the cells for 5-10 min by continuous perfusion.
5. Wash cells for 5 - 10 min by continuous perfusion.
6. For cell selection use a minimal amount of laser illumination, high scan rates (2-4 Hz), a small image size, and a high gain.
7. CAUTION: For the selection of Fluo5N-labeled cells don't use excess of light or direct illumination with epifluorescent light. Bright illumination of Fluo5N/Ca²⁺ complexes causes the complete loss of ER-specific fluorescence within 2-4 sec (Figure 5).
8. Set up the microscope according to the planned experiment. For orientation, representative settings for the imaging with different TED vectors are given in Table 1. For inverted laser scanning confocal microscopy, we use an Olympus IX81 microscope combined with a Fluoview 1000 confocal system that is equipped with diode lasers (473 nm, 15 mW; 559 nm, 20 mW) and a spectral detector system.
9. At the beginning of the experiment document the cells of interest by high-resolution x,y-z image stacks.
10. Perform x,y-t imaging to monitor ER calcium dynamics under the specific experimental conditions. Typical settings for our system are listed in Table 2.
11. Monitor changes in ER calcium under continuous perfusion with imaging solution. Typical buffer exchange rates are exemplarily: (a) cell washing and store-depletion by SERCA block: 1.5 ml/min; (b) ATP/DHPG stimulation: 3 ml/min.
12. Acquire digital images preferentially with 12-bit (4096 grey values). For parallel imaging of Fluo5N/Ca²⁺ and RFP, 8-bit images (256 grey values) may rescue your system from data overflow.
13. Store live cell time series images (x,y-t) or 3D image stacks (x,y-z) (for cellular distribution of Fluo5N/Ca²⁺ complexes) in an ImageJ compatible format (e.g. tiff).

8. Image Processing and Data Analysis

1. Open the image stack in the ImageJ program. In case of multicolor imaging, split the RGB channels when asked by ImageJ.
2. Identify your region(s) of interest (ROI) by a careful examination of the image stacks (e.g. with help of an intensity vs. time plot)
3. Use the Time-Series Analyzer plugin to read out the average pixel intensity in a ROI (F_raw). Depending on purpose, draw ROIs around either the complete ER or small regions of the ER e.g. ER of dendrites of peripheral cell regions. Also include 1-3 ROI in areas without cells for background subtraction.
4. Calculate the average background fluorescence (F_b) by calculating the mean fluorescence value of the background ROI and subtract the background value F_b from the F_raw values to get the F_roi value.
5. Calculate F₀, which is the basal fluorescence of the cells, by calculating the mean value of ten to 30 fluorescent values for each ROI in a resting state.
6. Calculate the background-corrected relative changes in fluorescence by ΔF/F₀ by the formula:

7. Present relative changes in fluorescence as a trace with ΔF/F₀ for the Y-axis and time for the X-axis.

Results

This protocol provides a non-disruptive approach for direct imaging of free ER calcium. Low-affinity synthetic Ca²⁺ indicators are released and trapped in the ER lumen with the help of an ER targeted, recombinant esterase enzyme activity. Improved loading of the Ca²⁺ indicator dyes to the ER lumen enables a direct and fast imaging of ER calcium store dynamics.

Cell types for TED

The feasibility of the method has been shown in the cell lines BHK21,...

Discussion

The advantages and disadvantages of the TED method have been discussed extensively in recent publications ^34,35. Compared to other methods described above, TED circumvents the problem of disrupting and perfusing the cells. Furthermore, two aspects need to be emphasized. The principle of TED for ER calcium imaging requires (1.) the targeted expression of an active carboxylesterase in the ER lumen with the help of TED vector constructs and (2.) the efficient and preferential release of low-affinity synthe...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
(S)-3,5-DHPG (Dihydroxyphenylglycine)	Tocris	0805
5';-ATP-Na2, ATP disodium salt hydrate	Sigma	A26209-1G
B-27 supplement,50x	Invitrogen	17504-044
Carbamoylcholine chloride (Charbachol)	Sigma	C4382-1g
Cyclopiazonic acid (CPA)	Ascent scientific	Asc-300
Dimethylsulfoxide (DMSO)	Sigma	D2650
DMEM with Glutamax	Invitrogen-Gibco	31966-047
Dulbecco's PBS without Ca/Mg 1x	PAA	H15-002
Fetal calf serum	Invitrogen-Gibco	10270-106
Fluo-5N-AM, 10 x 50 μg	Invitrogen	F14204
Glutamate	Ascent scientific	Asc-049
Glutamax	Invitrogen	35050-038
Ham's F12 nutrients mixture	Invitrogen-Gibco	21765-029
Hank's BSS(1x) without Ca,without Mg, with Phenol Red, HBSS	PAA Laboratories	H15-010
Horse serum	SHD3250YK	Linearis
Ionomycin Ca2+ salt	Ascent scientific	Asc-116
murine EGF	PreproTech	315-09
N2 supplement 100x	Invitrogen	17502-048
Neurobasal medium	Invitrogen-Gibco	21103-049
Pluronic F127, low UV absorbance,2g	Invitrogen	P6867
Poly-DL-ornithine hydrobromide PORN	Sigma	P8638
Poly-D-lysine hydrobromide	Sigma	P7280
Poly-L-Lysin	Sigma	P2636
Thapsigargin	Calbiochem	586005-1mg
TryLE Express	Invitrogen	12605-028
Trypsin	Worthington	LS-003707
Trypsininhibitor from Glycine MAX (soybean)	Sigma	T6522
Materials
Confocal system: inverted laser scanning microscope: FV1000 with IX81	Olympus		user-specific configuration
Confocal system: laser combiner FV10 and diode lasers (405, 473, 559, 635)	Olympus		user-specific configuration
Confocal system: scan head FV10-SPD with spectraldetector	Olympus		user-specific configuration
Heater controller TC-344B	Warner Instruments	64-0101	to control in line solution heater
NIH ImageJ software	WS Rasband, ImageJ, US National Institutes of Health, http://rsb.info.nih.gov/ij/, 1997--2006
Objective: UAPON20XW340 NA 0,7 WD 0,35	Olympus	N2709100
Objective: UPLFLN40XO	Olympus	N1478700
Objective: UPLSAPO60XO/1.35, WD=0.15	Olympus	N1480700
Perfusion chamber, inverse	custom-made
Perfusion chamber, RC-49FS	Warner Instruments	W4 64-1709
Perfusion tube: PT-49	Warner Instruments	64-1730	for perfusion
Peristaltic pump MiniPlus 3 (4 channels)	Gilson	n.a.	perfusion pump
Single inline solution heater SH-27B	Warner Instruments	64-0102	controlled by heater controller
Suction tubes: ST3R	Warner Instruments	64-1407	for perfusion
Heating insert: Tempocontrol 37-2 digital 2-channel	Pecon	n.a.