Dissection of Local Ca²⁺ Signals in Cultured Cells by Membrane-targeted Ca²⁺ Indicators

The dissection of Calcium signals at a sub-cellular resolution, is one of the most important steps for decoding intra-cellular calcium signals that determine the output biological phenomenon. This protocol describes a new calcium imaging method that enables the monitoring of the very moment of the calcium influx and calcium release. This protocol is applicable to all cell types that allows the expression of genetically encoded calcium indicators.

We believe that our method has the potential to be expanded to the dissection of calcium signals at the sub-cellular resolution in living animals in lab culture. Helping to demonstrate the procedure will be Matsumi Hirose, a technician from our laboratory. To begin this procedure, place 18 millimeter diameter glass cover slip in each well of a 12 well plate.

Use sterilized water, and prepare 12.5 milliliters of a 0.4%PEI solution for each 12 well plate being used. Add one milliliter of the PEI solution to each well, and ensure that there are no bubbles underneath the cover slips. Incubate over night in a carbon dioxide incubator at 37 degrees Celsius.

The next day, use an aspirator to remove the PEI solution. Add one milliliter of sterilized water to each well and shake the plate so that the PEI solution between the cover slip and the plate is washed out thoroughly. Repeat this wash process two additional times with fresh sterilized water.

After the final wash, aspirate the water from each well completely. Inside the hood, use an ultraviolet light to dry and sterilize the cover slips for at least 15 minutes. The PEI coated dish can be stored at four degrees Celsius for up to two months.

When ready to proceed, illuminate the dishes with the ultraviolet light for 15 minutes, just before use. Add five milliliters of sterile distilled water in the space between the wells to prevent evaporation of the culture medium. First, wash the cortices with incubation saline.

Then, incubate the cortices with Trypsin and DNAse in incubation saline for five minutes at 37 degrees Celsius. Then, wash the cortices three times with ice cold incubation saline. Remove the supernatant and add two milliliters of plating medium supplemented with 150 microliters of DNAse I stock.

Dissociate the cells by pipetting twenty times or less and filter the cells through a cell strainer with a pore size of 70 micrometers. Next, wash the cell strainer with 20 milliliters of the plating medium. For the preparation of frozen cell stock, wash the cells with 20 milliliters of the wash medium.

Dilute the cortical cells with plating medium to a density of 140 000 viable cells per milliliter. Then, add one milliliter of the diluted cell suspension to the PEI coated cover slips in the 12 well culture plates. Maintain the cells at 37 degrees Celsius in a carbon dioxide incubator for two to three days.

When the cells are stable, two to three days after plating, change the culture medium to the maintenance medium. For the preparation of frozen cell stock, spin down the cell, using a swing rotor to centrifuge the cells at 187 times G for three minutes. Aspirate the supernatant and add the cryopreservation medium, kept at four degrees Celsius, to obtain a cell density of 10 million cells per milliliter.

Aliquot one milliliter of the cell suspension into cryogenic tubes. Place the tubes into a cell freezing container with a freezing rate of minus one degrees Celsius per minute, until a temperature of minus 80 degrees Celsius is reached. Transfer the freezing container to a freezer at minus 80 degrees Celsius.

To revive the frozen cells, pre-warm the wash medium and the maintenance medium for frozen cortical cells. Next, thaw the frozen cells rapidly in a water bath at 37 degrees Celsius. Dilute the cells gently with pre-warmed wash medium and centrifuge with a swing rotor at 187 times G for three minutes.

Re-suspend the pellet in one milliliter of wash medium and measure the viable cell density. Then, dilute the cells with the maintenance medium for frozen cortical cells, to yield a cell density of 300 000 cells per milliliter. Cede one milliliter of this cell suspension into the wells of the PEI coated 12 well plates.

For transfection three to eight days after plating, label two tubes, one for plasma DNA and the other for the transfection reagent. Add 50 microliters of reduced serum medium per well, to each tube. Add 0.5 micrograms of plasma DNA per cover slip and one microliter of supplement accompanied by transfection reagent for neurons, per well to the plasma DNA tube.

For the co-transfection of Lck-RCaMP2 and OER-GCaMP6f, mix 0.5 micrograms of each plasmid and one microliter of supplement in 50 microliters of reduced serum medium per well. Next, add one microliter of transfection reagent per well to the transfection reagent tube. Vortex both tubes for one to two seconds.

Then, add the transfection reagent mixture to the DNA mixture. Gently pipette to mix and incubate at room temperature for five minutes. Load this mixture onto the cells in a drop-wise manner.

Incubate in a carbon dioxide incubator for two to three days until the marker proteins are expressed. Mix L and O to make the AAC infection. Add three microliters of AAC per well to the mixed neuron-astrocyte culture.

Gently rock the dish to mix. Maintain the culture for one to two weeks until the GECIs are expressed. First mount the cover slip containing the cells transfected with Lck-RCaMP2 and OER-CaMPG6f into the recording chamber of the microscope.

Add 400 microliters of the imaging medium and place a lid on top of the chamber. Choose the filter set for GCaMP6f and the light source. Locate the astrocytes expressing OER-GCaMP6f.

Next, choose a filter set and light source for RCaMP2 and confirm whether Lck-RCaMP2 is expressed in the same astrocytes. Record time lapse images of Lck-RCaMP2 at two Hertz for two minutes and save the imaging data. After this, change the filter set back to that for GCaMP6f.

Record time lapse images of OER-GCaMP6f at two Hertz for two minutes and save the imaging data. In this study, Lck-RCaMP2 and OER-GCaMP6f are expressed in HeLa cells and both signals were recorded simultaneously using image splitting optics, 24 hours after transfection. Upon the addition of Histamine, the signal intensity of Lck-RCaMP2 and OER-GCaMP6f increased.

The time courses of calcium ion elevation reported by Lck-RCaMP2 and OER-GCaMP6f are compared in the same regions of interest. Both sensors report an oscillation-like calcium ion elevation. And both show the same time course in two of the five cells examined.

However, the calcium ion elevations shown by Lck-RCaMP2 remain at the higher level than OER-GCaMP6f in the other cells. These results indicate that calcium ion elevation is prolonged in the vicinity of the plasma membrane, while it is terminated earlier around the ER, which is the source of this calcium ion signal induced by Histamine stimulation. Spontaneous calcium ion signals from astrocytes in the neuron-astrocyte mixed culture from rat hippocampi and cortices are shown by Lck-RCaMP2 and OER-GCaMP6f.

Spontaneous calcium ion elevations are visible only at the astrocytic process, not at the cell body which is consistent with previous reports. Spontaneous calcium ion elevations by Lck-GCaMP6f in immature rat hippocampal neurons are seen at two Hertz. The time courses for five different regions of interest suggest that these elevations are locally confined to the sub-cellular domains.

Mature mouse hippocampal neurons infected with OER-GCaMP6f-expression AAV vectors show calcium ion responses in response to the addition of DHPG. For calcium imaging, the excitation lumination power is the most critical factor. Please keep the excitation light as low as possible to avoid photo-bleaching and photo-toxicity.

The source of the calcium signals can further be confirmed by Pomakorzi using specific inhibitors to calcium channels. Decoding calcium signals to evoke specific output has been a fundamental biological question. This calcium imaging technique paves the way to describe the diversity of intra-cellular calcium signals.