Fluo 3 am

The effects of the pCBs on intracellular Ca²⁺ release that was induced by TRPV1 activation was determined using the selective intracellular fluorescent probe Fluo-3 AM (Molecular probes, Eugene, OR, USA). The HaCaT cells (1 × 10⁶ cells) were loaded for 15 min at 25 °C with 4 µM of Fluo-3 AM, which contained 0.02% Pluronic F-127 (Molecular probes) in an OPTIMEM medium, and then they were washed in the Tyrode’s buffer (145 mM of NaCl, 2.5 mM of KCl, 1.5 mM of CaCl₂, 1.2 mM of MgCl₂, 10 mM of D-glucose and 10 mM of HEPES; pH 7.4), resuspended in 2 mL of the Tyrode’s buffer and transferred into the quartz cuvette of the LS50B spectrofluorometer (Perkin Elmer, Waltham, MA, USA). Fluorescence was measured at 25 °C (excitation at λ = 488 nm; emission at λ = 516 nm) from the HaCaT cells, which had been pre-incubated with each pCB (6.0 µM of CBG, 4.0 of µM CBC, 9.0 µM of THCV and 13.0 µM of CBGA) and then stimulated with the selective TRPV1 agonist capsaicin (1 µM), as reported in [64 (link)]. The TRPV1-mediated intracellular Ca²⁺ elevation was expressed as fluorescence intensity (arbitrary units, AU) per 10⁶ cells.

For Fluo-3/AM staining, fresh pistils were excised with a razor and immediately placed in 10 mM MES buffer or 10 mM MES buffer containing 20 μM Fluo-3/AM (Molecular Probe, USA). After 5 h incubation at 4°C in the dark, the samples were washed with fresh Fluo-3/AM-free MES buffer before microscopic examination. Fluorescence from the pistils loaded with Fluo-3/AM was detected at 488 nm (excitation) and 520 nm (detection) under a laser scanning confocal microscope (ZEISS LSM 700).
For YC3.6 fluorescence observation, the UBQ10::YC3.6 transgenic plants [38 (link)] were crossed with the wild type 9311 and the sss1-D mutant, respectively. The pistils of the wild type plants and the homozygous mutants in the F₂ populations were excised with a razor and observed directly at 458 nm (excitation) and 525 nm (detection) under a laser scanning confocal microscope (ZEISS LSM 700). ZEN microscope and imaging software were used to measure the mean fluorescence intensity.

Oocytes from different treatment groups at 0, 8, 12, and 24 h during IVM were washed three times with a washing medium [DPBS with 3 mg/ml of bovine serum albumin (BSA)]; after which, they were incubated in a washing medium with 5 μM Fluo-3/AM (Invitrogen/Molecular Probes, Carlsbad, CA, United States) for 30–40 min in the dark at 39°C in 5% CO₂ and humidified air and washed three–five times to remove the Fluo-3/AM. Oocytes were allocated to 6-μl drops of DPBS under mineral oil and were ultimately observed using a Live Cell Imaging System (Nikon A1, Nikon, Tokyo, Japan). The [Ca²⁺]_i of oocytes was detected 10 times at intervals of 20 s using the fluorescence intensity of the laser at 488 nm (F488), and results were analyzed with software from NIS-Elements (Nikon, Tokyo, Japan).

Calcium imaging was performed with Fluo-3/AM (Invitrogen, Carlsbad, CA, USA) as described previously (9 (link)). Briefly, the intracellular calcium levels were determined using a fluorescence microscope (ECLIPSE Ti-U; Nikon, Tokyo, Japan; and Leica DMi8 inverted microscope; Leica Microsystems Ltd., Wetzlar, Germany) with the calcium-specific fluorescent dye, Fluo-3/AM (5 µM; Invitrogen, Carlsbad, CA, USA). Fluo-3/AM was mixed with 0.1% F127 in normal buffer solution (NBS: 140 mM NaCl, 5 mM KCl, 2 mM CaCl₂/EDTA, 0.5 mM MgCl₂, 10 mM glucose, and 5.5 mM HEPES, adjusted to pH 7.4), and loaded into the cells by incubating for 60 min at 37°C. After 60 min of incubation, the cells were washed twice with NBS and replaced with fresh NBS. Following treatment with the compounds, the alterations in the fluorescent images were recorded on a computer connected to the microscope. The excitation and emission wavelengths were 488 nm and 515 nm, respectively. The intracellular calcium levels were expressed as the F/F₀ ratio, where F indicates the fluorescence intensity of the region of interest at a certain time point, and F₀ indicates the initial fluorescence intensity at 0 s. Image analysis was performed using the ImageJ software (NIH), with custom-made scripts for semi-automatic cell counting, calculation of the F/F₀ ratio, and image production.

Fluo‐3/AM (Invitrogen, Carlsbad, CA, USA), a fluorescent indicator, was used to measure intracellular calcium levels and calcium image. To assess intracellular calcium levels, cells (1 × 10⁴ cells per well) were seeded in a black 96‐well plate (Greiner Bio‐One, Kremsmunster, Austria). Subsequently, cells were incubated with 10 µм Fluo‐3/AM for 1 h. Subsequently, the CAP patch was treated for 5‐ or 10‐min. The fluorescence intensity was detected at an excitation wavelength of 485 nm and an emission wavelength of 520 nm immediately. Intracellular calcium levels were compared to those of untreated cells, which were set at a value of one relative fluorescent unit. To investigate to Ca²⁺ image in keratinocytes, cells (1 × 10³ cells per well) were seeded in ibidi µ‐Slide 8‐well high glass‐bottom plates: # 1.5H Schott glass. After 24 h, cells were treated with 10 µм Fluo‐3/AM (Invitrogen) for 1 h in 37 °C. Subsequently, CAP patch treatment was performed for 5 or 10 min. Next, the cells were incubated with DAPI for 2 min. Images were acquired using a Zeiss confocal laser scanning microscope (Zeiss microsystems, Germany) at 400× magnification.

To detect intracellular ROS and NO, fungal mycelia were harvested at 0, 2, 4, 24, 48 h after incubation in glucose or Avicel medium. The harvested mycelia were stained with 20 μM 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA, Themo Fisher, Waltham, MA, USA) and 20 μM 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM DA, Themo Fisher) for intracellular ROS and NO, respectively, at 25 °C in the dark for 1 h.
Intracellular Ca²⁺ was detected using the fluorescent dye Fluo3-AM (Invitrogen, Carlsbad, CA, USA). Fungal mycelia were harvested at 0, 2, 4, 24, 48 h after incubation in glucose or Avicel medium and then stained with 5 μM of Fluo3-AM (Invitrogen) at 25 °C in the dark for 1 h.
After incubation, all stained samples were washed with 1× PBS and then imaged using a FV-100 MPE spectral confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan) under wavelengths corresponding to each fluorescent dye: 488/525 nm (excitation/emission) for H₂DCFDA, 495/515 nm (ex./em.) for DAFFMDA, 506/526 nm (ex./em.) for Fluo3-AM.

To visualize Ca²⁺ levels, D3(NMP-like) cells or D8 NPs (200K cells/cm²) were differentiated as described in Fig. 1B and incubated in a mixture of Fluo3AM (Invitrogen; stock 1 mM in DMSO, delivered to cells 1 μM) at 37°C for 30 min, rinsed with neurobasal medium (Gibco) supplemented as appropriate for D3 or D8 and left to recover for 1 h. Fluo3AM was then excited at 488 nm and the fluorescence generated was imaged by Deltavision Core microscope system in a WeatherStation environmental chamber maintained at 37°C. The D3(NMP-like) and D8 NP medium was buffered with a 5% CO₂/95% air mix and maintained in a humid chamber. Images were acquired using an Olympus 20×1.30 NA objective using a Xenon light source and a CoolSnap HQ2 cooled CCD camera (Photometrics). Images were deconvolved and maximum intensity projections of z-stacks were made using SoftWorx imaging software (Applied Precision). To provide a positive control for response to calcium influx, D3(NMP-like) and D8 NP cells were incubated with A23187 (Sigma C7522) 10 μg/ml in 0.1% DMSO in neurobasal medium) at 37°C for 20 min, rinsed, incubated in with Fluo3AM for 30 min and then rinsed in neurobasal medium. The fluorescence generated was imaged as above. The raw data were then quantified using ImageJ plugin Heatmap Histogram. Data and statistical analyses are presented in Fig. S7 and its legend.