2.1.

Chemicals

The cAMP plasmid, cAMP reagent stock solution, and FuGENE HD transfection reagent were obtained from Promega (Madison, Wisconsin). All other chemicals, unless otherwise specified, were obtained from Sigma (St. Louis, Missouri).

2.2.

Cell Culture and Transfection

HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C under humidified air containing 5% ${\mathrm{CO}}_2$. The experiment of HEK293T cells transfection was performed according to the cAMP assay technical manual. In brief, HEK293T cells were harvested and suspended at a density of $1.5\times {10}^5\text{cells}/\mathrm{ml}$ in growth medium (90% DMEM and 10% FBS) and were seeded into 96-well flat bottom plates by $100\mu \mathrm{l}/\text{well}$. Thus, the number of cells in the plate is $1.5\times {10}^4\text{cells}/\text{well}$. The plates were placed into a 37°C culture incubator with 5% ${\mathrm{CO}}_2$ overnight. The cAMP plasmid was diluted to a final concentration of $12.5\mathrm{ng}/\mu \mathrm{l}$ in Opti-MEM I reduced-serum medium (Invitrogen). Then, FuGENE HD transfection reagent ($6\mu \mathrm{l}$) was added to the diluted cAMP plasmid ($160\mu \mathrm{l}$) and mixed carefully by gentle pipetting. The complex ($166\mu \mathrm{l}$) was sufficient for 20 wells. After incubating for 15 min at room temperature, $8\mu \mathrm{l}$ of complex per well was added to 96-well plates and gently mixed without disturbing the cell monolayer. The 96-well plates were incubated for 24 h in a 37°C culture incubator with 5% ${\mathrm{CO}}_2$. The medium in 96-well plates was removed carefully and replaced with $100\text{-}\mu \mathrm{l}$ equilibration medium. The equilibration medium contains 88% ${\mathrm{CO}}_2$-independent medium (Invitrogen), 10% FBS, and 2% cAMP reagent stock solution. The 96-well plates were incubated for 2 h at room temperature or until a steady-state basal signal was obtained.

2.3.

Laser Irradiation

Three LQC laser diode modules (658, 785, and 830 nm, Newport Corporation, Irvine, California) were chosen as the light sources for LLLI. The laser beam was expanded by a lens and then reflected by a mirror to irradiate the cells in 96-well plates. The laser output was measured before and after laser irradiation by a laser power meter (Coherent, Wilsonville, Oregon) to check the stability of the laser output. The laser power densities on the irradiation surface were 20, 10, and $40\mathrm{mW}/{\mathrm{cm}}^2$ for 658-, 785-, and 830-nm laser diode modules, respectively. Different light doses were obtained by adjusting the irradiation duration. Laser irradiation was performed in a dark room at room temperature.

2.4.

Measurement of cAMP Level

A microplate fluorescence reader (Mithras LB940, Berthold, Germany) was utilized to measure the luminescence of cAMP reagent, which indicates the level of cAMP in living cells. The counting time was set as 1 s for obtaining a good signal-to-noise ratio. Adenosine receptors’ antagonists and other reagents were automatically injected into each well with injectors.

2.5.

Detection of Adenosine Receptors Using Laser Scanning Confocal Microscope

HEK293T cells were fixed with 4% paraformaldehyde for 1 h at room temperature. After being washed, cells were permeabilized with 0.3% Triton X-100 in 0.1 M phosphate buffer for 10 min. Cells were blocked in normal goat serum for 30 min and incubated with primary antibodies ($1:20$) overnight at 4°C. Then, cells were washed and incubated with Fluorescein Isothiocyanate-conjugated secondary antibodies ($1:50$) for 2 h at room temperature. At last, cells were mounted with Ultra Cruz TM Mounting Medium (sc-24941, Santa Cruz) and imaged with a laser scanning confocal microscope (Leica SP8, Germany).

2.6.

Measurement of the Temperature Distribution Induced by Laser Irradiation

The temperature distribution was measured with an infrared camera (Varioscan 3021, Germany). The temperature resolution is 0.03 K.

3.1.

Characteristics of the cAMP Reagent

To confirm the transfection and to obtain the cAMP reagent’s characteristics, we continuously monitored the cAMP luminescence signals of the blank control group (without transfection), control group (transfection), and forskolin group (transfection and adding forskolin to increase the cAMP level). There was no luminescence signal in the blank control group due to no transfection (data not shown). The luminescence signal of the control and forskolin groups was normalized by the initial value to reduce the influence of differences among wells. It is interesting that the normalized luminescence intensity of the control group increased for a while and then decreased gradually, as shown in Fig. 1. This is due to the differences of temperature between the environment and the microplate fluorescence reader. An increasing in temperature can decrease the luminescence intensity of the cAMP reagent, because the distance between the donor and acceptor will increase when the temperature rises. The temperature in the microplate fluorescence reader was higher than the room temperature. Therefore, the normalized luminescence intensity decreased when the 96-well microplate was placed in the microplate fluorescence reader. We noted that the change of the luminescence intensity with temperature was reversible (data not shown). To verify the validity of the measurement, further forskolin, which can activate adenylate cyclase to increase the cAMP level, was automatically injected into each well. The normalized luminescence intensity increased rapidly and up to sixfold after the injection of $10\text{-}\mu \mathrm{M}$ forskolin. These indicated that the transfection experiment was carried out in the correct way and that the system was sensitive for measuring the cAMP level in living cells.

Fig. 1

The normalized luminescence intensity changed with time. $I_i$ represents the initial luminescence intensity. Forskolin was injected into wells by injector automatically. The final concentration of forskolin was $10\mu \mathrm{M}$. The results were presented as the $\text{mean}\pm \mathrm{SD}$, $n=6$.

3.2.

Temperature Distribution Induced by Laser Irradiation

The cAMP reagent is temperature sensitive. To check the contribution of the laser irradiation on temperature change, a high-resolution infrared camera was used to measure the temperature distribution. The temperature at the center of the irradiation area, indicated by “+” in Fig. 2, increased gradually during laser irradiation. However, the maximum increment of the temperature was only 0.6 K [Fig. 2(b)] after being irradiated for 3 min by $40\mathrm{mW}/{\mathrm{cm}}^2$, 830-nm laser light. Moreover, the temperature decreased when irradiation was stopped. It returned to the initial value quickly [Fig. 2(d)]. Therefore, the contribution of LLLI on the temperature variation could be neglected.

Fig. 2

Temperature variation induced by laser irradiation: (a) before laser irradiation; (b) after irradiating for 3 min; (c) 1 min after irradiation was stopped; and (d) 3 min after irradiation was stopped. Laser parameters: 830 nm and $40\mathrm{mW}/{\mathrm{cm}}^2$. + indicates the irradiation spot, and * indicates the nonirradiation area.

3.3.

cAMP Dynamics After Laser Irradiation

In order to study the effect of laser irradiation on the dynamics of cAMP, we recorded the cAMP level every 30 s after the laser irradiation. The laser irradiation experiments were performed using an 830-nm laser diode providing a $40\mathrm{mW}/{\mathrm{cm}}^2$ power density. Two groups of samples were irradiated: one for 30 s and another for 60 s. A control group did not receive any laser irradiation, and its cAMP level was also measured at the corresponding time points for the other two groups. All the samples (all three groups) were put on the same 96-well plate, so that they were always at the same temperature and humidity conditions during the experiment. After the irradiation, the samples were placed in the microplate fluorescence reader and the cAMP signal levels were then monitored continuously as a function of time. The results are shown in Fig. 3. The normalized luminescence intensity of the control group decreased gradually similar to what is shown in Fig. 1. Unlike the control group, the cellular cAMP level of the irradiation groups remained largely unchanged. This indicates that the LLLI could raise the cAMP level, which is consistent with previous reports.^26,29

Fig. 3

Laser irradiation inhibited the cAMP level. $I_i$ represents the initial luminescence intensity. The wavelength of the laser is 830 nm, and the power density is $40\mathrm{mW}/{\mathrm{cm}}^2$. Irradiation duration was 30 and 60 s, respectively. The results were presented as the $\text{mean}\pm \mathrm{SD}$, $n=6$.

The increase of cAMP level may be due to the increase of ATP induced by LLLI. Numerous studies demonstrated that LLLI can enhance the ATP synthesis.^40–44 ATP can be hydrolyzed sequentially to adenosine diphosphate (ADP), adenosine monophosphate (AMP), and finally adenosine.⁴⁵ Increasing ATP and adenosine could raise the cAMP level indirectly by activating the adenylate cyclase.⁴⁵

For studying the effect of laser wavelength on the modulation of the cAMP level, 658-, 785-, and 830-nm lasers were used to irradiate the cells. As the power densities are different between the three lasers, which are 20, 10, and $40\mathrm{mW}/{\mathrm{cm}}^2$, to obtain the same light dose, we set the irradiation duration as 1, 2, and 0.5 min, respectively. The normalized luminescence intensities after laser irradiation were compared to evaluate the wavelength effect. Figure 4 clearly shows that the normalized luminescence intensities at 20 min after laser irradiation were greater than that of control group (*$p<0.01$). However, there were no significant differences among the three laser irradiation groups (**$p>0.05$).

Fig. 4

The effect of laser wavelength on the modulation of cAMP level. There were significant differences between the control group and any of the three laser irradiation groups for the cellular cAMP level at 20 min after laser irradiation (*$p<0.01$). There were no significant differences among the three laser irradiation groups (**$p>0.05$). The light doses of the three lasers are the same, 456 mJ. The results were presented as the $\text{mean}\pm \mathrm{SD}$, $n=6$.

3.4.

Role of Adenosine Receptors in the Modulation of cAMP

There are four subtypes of adenosine receptors, which are ${\mathrm{A}}_1\mathrm{R}$, ${\mathrm{A}}_{2\mathrm{a}}\mathrm{R}$, ${\mathrm{A}}_{2\mathrm{b}}\mathrm{R}$, and ${\mathrm{A}}_3\mathrm{R}$. Immunofluorescence was used to check the expressions of the four subtypes of adenosine receptors on HEK293T cells. Figure 5 shows that all the four subtypes of adenosine receptors expressed on HEK293T cells. However, their expressions were different. ${\mathrm{A}}_{2\mathrm{a}}\mathrm{R}$ had the strongest expression, whereas ${\mathrm{A}}_1\mathrm{R}$ had the weakest expression. ${\mathrm{A}}_{2\mathrm{b}}\mathrm{R}$ and ${\mathrm{A}}_3\mathrm{R}$ had similar expressions.

Fig. 5

Images of adenosine receptors (${\mathrm{A}}_1\mathrm{R}$, ${\mathrm{A}}_{2\mathrm{a}}\mathrm{R}$, ${\mathrm{A}}_{2\mathrm{b}}\mathrm{R}$, and ${\mathrm{A}}_3\mathrm{R}$) on HEK293T cells by confocal microscopy. $\text{Scale bar}=10\mu \mathrm{m}$.

To explore the role of adenosine receptors on the increase of cAMP level induced by laser irradiation, we applied the ${\mathrm{A}}_1\mathrm{R}$, ${\mathrm{A}}_{2\mathrm{a}}\mathrm{R}$, and ${\mathrm{A}}_3\mathrm{R}$ antagonists before laser irradiation. We first studied the effects of the receptor antagonists alone on the cAMP level. ${\mathrm{A}}_1\mathrm{R}$ antagonist (DPCPX), ${\mathrm{A}}_{2\mathrm{a}}\mathrm{R}$ antagonist (ZM 241385), and ${\mathrm{A}}_3\mathrm{R}$ antagonist (VUF 5574) were automatically injected into the 96-well plate. There were no significant differences between the VUF 5574 group and the control group at the end of measurements, while there were a few differences between the DPCPX group, ZM 241385 group, and the control group, as shown in Fig. 6.

Fig. 6

Effect of adenosine receptors’ antagonists on the cellular cAMP level. There was no difference between the control group and the VUF 5574 group at the end of measurement. There were differences between the control group and DPCPX and ZM 241385 groups. The results were presented as the $\mathrm{mean}\pm \mathrm{SD}$, $n=6$. DPCPX, ${\mathrm{A}}_1\mathrm{R}$ antagonist; ZM 241385, ${\mathrm{A}}_{2\mathrm{a}}\mathrm{R}$ antagonist; and VUF 5574, ${\mathrm{A}}_3\mathrm{R}$ antagonist.

Then, the cells applied with adenosine receptor antagonists for 20 min were irradiated by an 830-nm laser for 3 min per well. The power density was $40\mathrm{mW}/{\mathrm{cm}}^2$. As shown in Fig. 7, laser irradiation could increase the cAMP level of the three groups of HEK293T cells injected with DPCPX, ZM 241385, and VUF 5574 compared with the control group. There were no differences between the groups of DPCPX and ZM 241385; however, laser irradiation increased the cAMP level of the VUF 5574 group more than DPCPX or ZM 241385 group. These results were in good agreement with the adenosine receptors expressions on HEK293T cells. The four subtypes of adenosine receptors are GPCRs with seven transmembrane domains. ${\mathrm{A}}_1\mathrm{R}$ and ${\mathrm{A}}_3\mathrm{R}$ preferably interact with members of the ${\mathrm{G}}_{\mathrm{i}}$ family and inactive adenylate cyclase to decrease the production of cAMP, whereas ${\mathrm{A}}_{2\mathrm{a}}\mathrm{R}$ and ${\mathrm{A}}_{2\mathrm{b}}\mathrm{R}$ are coupled to the ${\mathrm{G}}_{\mathrm{s}}$ family and stimulate adenylate cyclase to increase the production of cAMP.⁴⁶ Since ${\mathrm{A}}_1\mathrm{R}$ was scarce in the HEK293T cells, when the ${\mathrm{A}}_3\mathrm{R}$ was blocked by the antagonists VUF 5574, the increasing adenosine induced by LLLI would activate the ${\mathrm{A}}_{2\mathrm{a}}\mathrm{R}$ and ${\mathrm{A}}_{2\mathrm{b}}\mathrm{R}$ to stimulate the adenylate cyclase to raise the cAMP level.

Fig. 7

Effects of adenosine receptors’ antagonists on the increase of the cAMP level in HEK293T cells irradiated by laser. The concentrations of these reagents were all $10\mu \mathrm{M}$. The results were presented as the $\mathrm{mean}\pm \mathrm{SD}$, $n=6$. DPCPX, ${\mathrm{A}}_1\mathrm{R}$ antagonist; ZM 241385, ${\mathrm{A}}_{2\mathrm{a}}\mathrm{R}$ antagonist; and VUF 5574, ${\mathrm{A}}_3\mathrm{R}$ antagonist.