Cell culture

hTERT ASC52telo (ATCC® SCRC-4000™) cells were cultured in Dulbecco's Modified Eagle Media (DMEM) (Sigma-Aldrich, D5796) with an addition of 10% fetal bovine serum (FBS Superior) (Biochrom, S0615), and the culture medium was supplemented with 1% antibiotics, comprising 0.5% Penicillin–Streptomycin (Sigma-Aldrich, P4333) and 0.5% Amphotericin B solution (Sigma-Aldrich, A2942). The cells were grown in a controlled environment at 37 °C with 5% CO2 and 85% humidity inside a Heracell™ 150i CO2 Incubator (Thermo Scientific™, 51026280). They were cultured in cell culture flasks from Corning® (Sigma, CLS430639/ CLS430641/ CLS431080). Once the cells reached full confluence, they were transitioned to a specialized neuronal differentiation medium containing the specified components: Indomethacin [200 µM] (I7378-10G, Merck/SIGMA), Insulin [5 µg/ml] (I9278-5ML, Merck/SIGMA), and 3-Isobutyl-1-methylxanthine (IBMX) [0.5 mM] (I5879-100MG, Merck/SIGMA).

Photobiomodulation

After the cells were cultured, approximately 100,000 cells were placed in 35 mm diameter petri dishes (Corning, 430165) with complete medium and left to incubate for 24 h before the irradiation process. The attached ADMSCs were subjected to two types of lasers: a NIR 825 nm Diode Laser (National Laser Centre of South Africa, SN 070900108) equipped with a 1000 mA LaserSource (ArroyoInstruments, 4210) and a G 525 nm Diode Laser (National Laser Centre of South Africa, EN 60825–1:2007) with a power source of 100–240 VAC and 47–63 Hz at 5A (OptoElectronics Tech.CO.,LTD). The laser's output power in milliwatts (mW) was measured using a FieldMate Laser Power Meter (Coherent, 1098297). The irradiation time was determined based on the energy output, using a High-Sensitivity Thermopile Sensor PM3 (Coherent, 1098336). The laser spot size was sufficient to cover the entire cell monolayer. Specific laser parameters are detailed in Table 1 for reference.

Table 1 Laser parameters

The cell cultures were segregated into three distinct experimental groups: 1) cells subjected to NIR irradiation at 825 nm with a dosage of 5 J/cm2, 2) cells subjected to G irradiation at 525 nm with a dosage of 5 J/cm2, and 3) cells treated with NIR at 825 nm followed immediately by G at 525 nm, both at a fluency of 5 J/cm2. Cells that were not exposed to laser treatment (0 J/cm2) were included as control samples. The duration of exposure for each laser wavelength was determined using the following formula:

$$Time (\text)=\frac^}^}$$

Characterization of markersFlow cytometry

Secondary antibody conjugation was used to label cultivated hTERT ASC52Telo (ATCC® SCRC-4000™) cells with a fluorescent marker that was subsequently observed through flow cytometry (BD 468 Biosciences, BD ACCURI C6 PLUS). According to the ATCC® guidelines, the CD44 marker was tested for. Cultivated cells were cultured with complete media in sterilized 35 mm diameter petri dishes (Corning, 430165) with a seeding density of 1 × 105. An incubation period of 24 h was used to adhere cells to the petri dish. Cells were washed three times with a cold washing solution (Azide/PBS/BSA: containing 0.01% w/v Sodium azide from Sigma-Aldrich, PBS, and 0.1% w/v BSA from Sigma-Aldrich). Afterward, the ADMSCs were chilled and treated with a blocking solution for 30 min (BSA/PBS: consisting of 10% w/v BSA in PBS). The cells were washed three times with the cold washing solution again. Next, the cells were exposed to the primary antibody, anti-CD44 (ThermoFisher, MA110225), which was diluted in a working solution (Azide/PBS/BSA/FBS: containing 0.01% w/v sodium azide from Sigma-Aldrich, PBS, 0.1% w/v BSA, and 2% FBS from Biochrom) for 30 min on ice. Subsequently, the cells were washed three times with the washing solution and then incubated with the necessary secondary fluorescent antibody, Cy5 goat anti-mouse (NovusBio, NB7602), for 30 min in the dark on ice. The washing step was repeated. Following this, the cells were fixed with 10% paraformaldehyde (P6148, Sigma-Aldrich) for 10 min. After fixation, the cells were washed three times with PBS and finally resuspended in PBS. The cells were promptly analyzed using a flow cytometer (Fl-4, Cy5 filter) from BD Biosciences (BD ACCURI C6 PLUS).

Biochemical analysisMorphology: inverted light microscopy

Morphological changes were examined and analyzed at 24 h, 48 h, and 7 days after laser treatment using inverted light microscopy (OLYMPUS CKX41). These observations were captured with a digital camera attached to the microscope (OLYMPUS, SC30), and the images were processed using the cellSens software.

Cell viability: trypan blue exclusion assay

A color exclusion assay was conducted to assess cell viability using the Trypan Blue Staining method (0.4%) from Invitrogen (T10282). The Countess Automated Cell Counter (ThermoFischer, AMQAX1000) was employed to visualize the cells and, subsequently, calculate the percentage of viable cells.

Proliferation: ATP luminescence assay

Cell proliferation was assessed using the ATP luminescence assay known as CellTiter-Glo® 2.0 (Promega, G9241). The luminescent intensity was quantified by the VICTOR3 Multilabel Plate Counter (PerkinElmer, HH3522019094), which measured the relative light units (RLUs).

Lactate dehydrogenase

The LDH cytotoxicity assay was employed to quantify the release of lactate dehydrogenase (LDH) using the CytoTox 96® non-radioactive cytotoxicity assay. This assay assesses the LDH released due to cell membrane damage by measuring the amount of LDH generated during the conversion of lactate into pyruvate. It is a colorimetric assay that produces a red by-product, and its reactivity depends on NADH. The measurement was carried out at a wavelength of 490 nm using a spectrophotometer.

Reactive oxygen species (ROS) detection – spectrophotometry

To measure ROS production and to determine whether cellular damage occurred due to stimulated ROS generation, the Image-IT® LIVE Green Reactive Oxygen Species Detection Kit (Life Technology I36007) was used. ROS, along with free radicals, are generated during cellular metabolism as part of redox reactions when cells are stimulated externally. When calcein and fluorescein levels are low, the presence of ROS is indicated. When the acetate groups of 2’, 7’-di-chlorofluorescein (DCF) and calcein are removed through intracellular esterases, oxidation occurs, and fluorescence is observed. Using this method, ROS was observed through spectroscopic means (VictorNivo).

Statistical analysis

Biochemical assays were carried out in triplicate (n = 3). Spectrophotometric investigations included the incorporation of a blank sample generated from the collected data. To analyze the data, statistical tests were employed: the Student's t-test for comparing two groups and one-way ANOVA for comparing all groups. These analyses were performed using the SigmaPlot program version 12. Morphological data was quantitatively assessed using ImageJ, which is a publicly accessible Java-based image processing system developed by the National Institute of Health in Bethesda, MD, USA. The data is presented as the mean ± standard error (SE). Statistical significance is indicated in tables and graphs as follows: P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***), and the standard error is visually represented using error bars.