F14201

For calcium staining in living cells, fluo 4 (F-14201, Invitrogen, Oregon, USA) was used. Five μl fluo 4 (50 μg fluo4 in 50 μl DMSO) were added to 1 ml medium of neurons and they were incubated for 45 min at 37 °C. Afterwards they were washed in 1x PBS and further incubated for 30 min. Nuclei were stained with Hoechst (H3570, Invitrogen). For cdh1 analysis in neurons, they were fixed in formaldehyde (4% in 1x PBS) and stained using anti-cdh1 (1:100, NBP2-15840, Novusbio) and Map2 (1:100, M9942, Sigma). Fluorescent secondary antibodies were used as above. Nuclei were stained with Hoechst (H3570, Invitrogen).

Immunopanned CalEx; Cnp-Cre OPCs were differentiated for two to three days for all calcium imaging experiments. To load 1 μM cell permeable calcium indicator, Fluo-4-AM (Invitrogen, F14201) 50 μg tube of lyophilized Fluo-4-AM stock was resuspended in 46 uL of sterile DMSO (1 mM concentration). 1.5 μL of Fluo-4-AM stock was added directly to cell media to prevent shearing of cells. Cells were incubated with Fluo-4-AM for 20 minutes and then media was completely changed to 1 mL DMEM-SATO Media (made with Fisher Scientific A1896701) for imaging.
Cells were imaged on an Opterra II Multipoint Swept Field Confocal outfitted with a humidified, temperature-controlled microscope enclosure (Okolab microscope enclosure, H201-Temperature Unit, CO2 controller, HM-Active Vibration Free Humidity Controller with humidity sensor and temperature-controlled tube). Imaging was performed using the 60x/1.2 NA water objective and Perfect Focus to prevent z-plane drift during imaging. Images were acquired every two seconds in 488 channel with 70 μM slit and 100 ms exposure time with 15% laser power. Calcium imaging data was analyzed using Fiji to select regions of interest and was analyzed to extract rate and amplitude measurements. All calcium imaging was performed on oligodendrocyte processes; soma events were relatively rare compared to process events. Data was analyzed blind to condition.