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1. Plating the cells
NOTE: Based on experience, about 100,000 viable cells per embryo are collected. 2–3-month-old timed pregnant mice typically have litter sizes of 8-10 embryos; therefore, a rough estimate for total yield of cells per timed pregnant mouse is approximately 1 million cells.
- Using a hemocytometer, perform a cell count and then dilute the suspension to 2,000 cells/µL using cell culture medium. Triturate briefly to mix.
- Remove coverslips with laminin solution from the incubator and aspirate the remaining laminin solution from the coated coverslips using a vacuum. Plate quickly to avoid the coverslips from drying completely. Pipette 100 µL (2.0 x 105 cells/coverslip) onto each coverslip and place Petri dishes into a 37 °C incubator for 1 h.
- Carefully add 3 mL of cell culture medium to each dish and place back into the 37 °C incubator. Preform half medium changes 2 times per week for 2 weeks.
2. Infection of cell culture at 14 (days in vitro) DIV with adeno-associated viral (AAV) vectors
- For each dish prepare 1 mL of serum free DMEM medium with 1 µL of hSyn-GCaMP6f AAV (1.0 x 1013 titer)
- Aspirate the cell culture medium from each dish and replace with 1 mL of serum free Dulbecco's Modified Eagle Medium (DMEM) containing hSyn-GCaMP6f. Place dishes back into the 37 °C incubator for 1 h.
- Aspirate the serum free medium containing AAVs and replace with 3 mL of cell culture medium. Place dishes back into the 37 °C incubator. We have found that 5-7 days of AAV infection allows for ideal levels of GCaMP expression. Continue to change medium every 2-3 days throughout this period of viral infection.
3. Live confocal Ca2+ imaging between 19-21 days in vitro (DIV)
NOTE: Imaging can be done between 5-7 days following viral infection. This is the ideal window to achieve visible expression of the fluorophore at levels which allow for detection of spontaneous Ca2+ activity.
- Preparation of recording buffers
- To make 1 L of HEPES recording buffer, add: 9.009 g of NaCl, 0.3728 g of KCl, 0.901 g of D-glucose, 2.381 g of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2 mL of 1 M CaCl2 stock solution, and 500 µL of 1 M MgCl2 stock solution to 800 mL of sterile distilled H2O. Bring the pH to 7.4 with NaOH. Bring to a final volume of 1 L.
- To make 200 mL of 20 µM glutamate recording buffer, dilute 40 µL of 100 mM glutamate stock solution into 200 mL of HEPES recording buffer described above.
- Confocal imaging
- Fill a sterile 35 mm Petri dish with 3 mL of recording buffer.
- Remove a 35 mm Petri dish with infected cultures from the 37 °C incubator. Using fine tip forceps, carefully grab the edge of one coverslip and transfer it quickly into the Petri dish filled with recording buffer. Place the remaining coverslip in medium back into the 37 °C incubator. Transport the dish with the recording buffer to the confocal microscope.
- Start the imaging software. Proceed to the next step while it initializes.
- Start the peristaltic pump and place the line into the recording buffer. Calibrate the speed of flow to be 2 mL/min.
- Transfer the infected coverslip from the 35 mm Petri dish into the recording bath.
- Using the 10x water immersion objective and bright-field (BF) light, find the plane of focus and look for a region with a high density of neuron cell bodies. Switch to the 40x water immersion objective and use BF light to refocus the sample.
- In the “Dyes list” window within FluoView select AlexaFluor 488 and apply it.
- AAV expressions can be variable; therefore, in order to prevent overexposure and photobleaching of the fluorophores, start with low HV and laser power settings. For the AlexaFluor 488 channel, set the high voltage (HV) to 500, the gain to 1x, and offset to 0. For the 488 laser line set the power to 5%. To increase the effective volume imaged in the z-plane, increase the pinhole size to 300 µm. Use the “focus x2” scanning option to optimally adjust emission signals to sub-saturation levels. From here, settings can be adjusted until ideal visibility of each channel is achieved.
NOTE: To accurately capture the full range of Ca2+ fluxes with GCaMP, adjust the baseline HV and laser power settings to allow for an increase in fluorescent intensity without oversaturating the detector. - Once microscope settings are optimized, move the stage to locate a region with multiple cells displaying spontaneous changes in GCaMP6f fluorescence and focus to the desired plane for imaging.
- Use the “Clip rect” tool to clip the imaging frame to a size that can achieve a frame interval of just under 1 second. This is necessary to set the imaging interval at 1 frame per second.
- Set the “Interval” window to a value of 1.0 and the “Num” window to 600.
NOTE: In order to deliver different recording buffers at the desired time point (300 s), it is important to calibrate the latency of the pump to deliver the new solution to the bath. This will be dependent on the solution perfusion rate (2 mL/min) and the length of the line used to pump solution. - To capture a t-series movie select the “Time” option and then use the “XYt” scanning option to begin imaging.
- Watch the imaging progress bar and move the line from the HEPES recording buffer into the 20 µM glutamate recording buffer at the appropriate time point (e.g., if the latency of the pump is calibrated to deliver solution at 60 s, move the line into the glutamate buffer at 240 frames in order to deliver glutamate at 300 s).
- When imaging is complete, select the Series Done button and save the finished t-series movie. Continue to perfuse 20 µM Glutamate for an additional 5 min, so that the cultured neurons have been exposed to glutamate for a total of 10 min. Repeat this process for each coverslip to be imaged.
- Following the additional 5 min exposure to 20 µM Glutamate, remove the coverslip from the bath and place back into the 35mm Petri dish containing recording buffer until the day of imaging is completed.