Missing part of laser article

By backspace
May 17, 2008

MATERIALS AND METHODS

Cell culture and preparation. Rat basophilic leukemia (RBL-2H3) cells were obtained from the cell bank of Shanghai Science Academe. Cells were grown in minimal essential medium (MEM) with Earle’s salts containing 10% fetal bovine serum, 2% L-glutamine (all from Gibco), in an incubator with a humidified atmosphere (5% CO2) at 37°C. Cells in exponential phase of growth were used.

Fluorescence measurements of intracellular Ca^sup 2+^ . The cells were seeded on the cover-slips in 12-well culture plates and allowed to attach for 12 h. Before the measurements, cells were washed with Hank’s buffer solution (KH^sub 2^PO^sub 4^ 0.06 g, NaCl 8.0 g, NaHCO^sub 3^ 0.35 g, KCl 0.4 g, glucose 1.0 g. Na^sub 2^HPO^sub 4^*H^sub 2^O 0.06 g. CaCl^sub 2^ 0.14 g. MgSO^sub 4^*7-H^sub 2^O 0.20 g, add H2O to 1000 mL), and then incubated with 10 µM fluo-3/AM (Dojindo, Tokyo, Japan) at 37°C for 30 min. After three washes, these stained cells were sealed on the slides for fluorescence microscopical measurements.

Fluorescence images of intracellular [Ca^sub 2+^ ]^sub i^ were made by confocal fluorescence microscopy (DSU, IX-71; Olympus, Japan). The blue light (460-490 nm) filtered from an attached xenon lamp was selected for excitation. Fluorescence signals were acquired by an attached CCD detector (Evolution QEi, monochrome; Media Cybernetics, CA) with 510-550 nm band-pass filter. A pinhole disk was assembled in the microscope with a 60×/1.20 water-immersion objective lens. Confocal fluorescence images were obtained with the resolution of 0.5 µm in the z-axis and processed with the Image-Pro Plus software (version 5.1; Media Cybernetics). With such a system, the distribution and amount of [Ca^sup 2+^]^sub i^ can be studied with a good resolution in three dimensions. After 1-min irradiation, a series of fluorescence images of [Ca^sup 2+^]^sub i^ were captured with the exposure time of 300 ms in every 10 s for 2 min. To minimize the phut o bleaching effect, an ND filter (12%) was used to decrease the intensity of excitation.

Measurements of histamine release. Histamine release from RBL-2H3 cells was measured by the method of fluorescence staining with the highly specific reagent o-phthalaldehyde (OPA) (Sigma, USA), as initially described by Shore et al. (19) and later improved by others (20-22).

Cells were seeded in 96-well flat bottom plates (5 × 10^sup 5^ cells/well). Before laser irradiation the MEM medium was replaced by Hank’s buffer to give the lowest background in fluorescence measurements.

After laser irradiation, the supernatant in each well was collected and incubated with OPA (0.1 mM, 300 µL) for 20 min. The fluorescence spectra and relative intensities of the supernatants were measured, respectively, by an F-2500 fluorescence spectrophotometer (Hitachi, Japan) with the excitation wavelength of 352 nm. The cell viability after laser irradiation was measured by the MTT assay.

Laser irradiation. To study laser-induced changes in [Ca^sup 2+^]^sub i^, a 405 nm laser (Coherent, CA) was introduced into the microscope in order to irradiate a single cell. The total laser power of 0.96 mW on the cell was measured with a power meter (laser-check; Coherent). The irradiation area was about 28 µm^sup 2^ (irradiation radius was about 3 µm) on the focus of the objective (60×). The irradiation time was 60 s.

To examine the effect of laser on histamine release, the 405 or 532 nm laser (solid laser; CrystaLaser) was used. The diameter of the beam spot on the cell dish was about 3 mm, and the irradiation power was adjusted by a polarizer from 0 to 10 mW according to the designed experiments. Cells were divided into different groups (three wells for each group) and irradiated with different light doses. One group with nonirradiation as a control was also included.

Immunocytochemical detection of TRPV4 in RBL-2H3 cells. The transient receptor potential vanilloid 4 (TRPV4) is a cation channel protein localized in the membranes of some cell lines, which can be activated by diverse physical and chemical stimuli to mediate Ca^sup 2+^ influx (23). To verify the presence of TRPV4 in RBL-2H3 cells, an immunocytochemical method was used. RBL-2H3 cells were seeded onto glass coverslips in cell dishes. After 16 h cells were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 20 min at room temperature followed by permeation with 0.1% Triton-X-100 for 15 min. The permeation was blocked with 10% normal goat serum in PBS for 30 min. The primary antibody used was rabbit anti-rat TRPV4 (Sigma) and diluted 1:150 in 1% blocking solution and applied for 1 h at 37°C. Then, secondary antibody (goat anti-rabbit IgG-FITC conjugated, diluted 1:400 in PBS [Sigma]) was applied for another 1 h at 37°C. Cells were imaged by the above fluorescence microscopic system with excitation at 460-490 nm and fluorescence capture at 510-540 nm. The nuclei of cells were stained with Hoechst 33342 and imaged at the region of 400-460 nm with an excitation filter of 320-360 nm.

Influence of Ruthenium red (RR) on histamine release. Ruthenium red is known to inhibit the function of TRPV4 and thus was used to examine the effect of TRPV4 on histamine release induced by laser in RBL-2H3 cells. The cell preparation and histamine assay were the same as described above. The cells were divided into four groups including nonirradiation control, laser irradiation alone, irradiation plus RR (3 µM) and RR (12 µM). The cell culture dish was irradiated with 10 mW laser (532 nm) using the beam spot of 3 mm diameter. The irradiation time was 10 min.

In some experiments with measurements of [Ca^sup 2+^]^sub i^ and histamine, the Ca^sup 2+^-free saline solution (pH 7.0) was used as comparison, as the Hank’s buffer contains a large amount of Ca^sup 2+^.

Statistical analysis. Each set of experiments was repeated at least three times. Fluorescence intensities at the peak wavelength of the OPA-histamine complex for each group were calculated and expressed as mean ± SEM. Statistical comparisons between treated groups and an untreated one (control) were determined by using one-way ANOVA and significant difference was accepted when P < 0.05.