Cells on 35 mm dishes (MatTek) were loaded with Fluo-4 (Thermo, F10471) or Fura-2 (Thermo, F1221). The dye was removed and 1 mL of HBSS with 20 mM HEPES, pH 7.4 (Thermo, 14170112) was added to each dish for 10 min prior to microscopy. A brightfield image was taken prior to cell stimulation and imaging. Fluo-4 microscopy was performed using Spinning Disk Confocal microscopy at 40X (PerkinElmer) with Volocity imaging software (Perkin Elmer). Cells were imaged at 488 nm. After a 10 sec baseline recording, cells were treated with controls or extracts for 90 sec and images were obtained every 1 sec. A waiting period of 2–5 min was performed between treatment of cells with another molecule to minimize neurons being desensitized.
For Fura-2 experiments, after dye loading, cells were placed in 1 mL of HBSS with 20 mM HEPES for 10 minutes prior to recording. Using an Olympus IX73 inverted microscope, data was acquired with MetaFluor Flourescence Ratio imaging software (Olympus) at excitation wavelengths of 340/380 nm and emission wavelength of 510 nm with image acquisition every 1 sec. A 100 second baseline was first recorded after which DMSO was added and recording was obtained for 300 sec. Next, compounds or extracts were added with continuous recording for 300 sec. Finally, 200 nM was capsaicin added with continuous recording for 300 sec.
For Fura-2 experiments, after dye loading, cells were placed in 1 mL of HBSS with 20 mM HEPES for 10 minutes prior to recording. Using an Olympus IX73 inverted microscope, data was acquired with MetaFluor Flourescence Ratio imaging software (Olympus) at excitation wavelengths of 340/380 nm and emission wavelength of 510 nm with image acquisition every 1 sec. A 100 second baseline was first recorded after which DMSO was added and recording was obtained for 300 sec. Next, compounds or extracts were added with continuous recording for 300 sec. Finally, 200 nM was capsaicin added with continuous recording for 300 sec.