Dataset of PNT1A and PC-3 cells - efect of FITC phototoxicity, quantitative phase imaging (1/2)

Part of article Feith, M,Vičar, T., Gumulec, J.,  Raudenská, M. Wingren, AG, Masařík, M., Balvan, J. Quantitative Phase Dynamics of Cancer Cell Populations Affected by Blue Light, Appl. Sci. 2020, 10

Increased exposition to blue light may induce many changes in cell behavior and significantly affect the critical characteristics of cells. Here we show that multimodal holographic microscopy (MHM) within advanced image analysis is capable of correctly distinguishing between changes in cell motility, cell dry mass, cell density, and cell death induced by blue light. We focused on the effect of blue light with a wavelength of 485 nm on morphological and dynamical parameters of four cell lines, malignant PC-3, A2780, G361 cell lines, and the benign PNT1A cell line. We used MHM with blue light doses 24 mJ/cm², 208 mJ/cm² and two kinds of expositions (500 and 1000 ms) to acquire real-time quantitative phase information about cellular parameters. It has been shown that specific doses of the blue light significantly influence cell motility, cell dry mass and cell density. These changes were often specific for the malignant status of tested cells. Blue light dose 208 mJ/cm² × 1000 ms affected malignant cell motility but did not change the motility of benign cell line PNT1A. This light dose also significantly decreased proliferation activity in all tested cell lines but was not so deleterious for benign cell line PNT1A as for malignant cells. Light dose 208 mJ/cm² × 1000 ms oppositely affected cell mass in A2780 and PC-3 cells and induced different types of cell death in A2780 and G361 cell lines. Cells obtained the least damage on lower doses of light with shorter time of exposition.

Materials and Methods 

Cell Lines

The PC-3, A2780, PNT1A, and G361 cell lines were purchased from HPA Culture Collections (Salisbury, UK). PC-3 prostate cancer cell line was derived from bone metastasis of a 4-grade prostatic adenocarcinoma of a 62-year-old Caucasian male  The A2780 cell line was derived from the ovarian carcinoma of a nontreated patient according to ECACC. PNT1A cell line was established from prostatic epithelial tissue of healthy 35-years old male and immortalized by plasmid transfection containing the SV40 genome with defective replication origin. The G361 cell line was established from a malignant melanoma of a 31-year-old male Caucasian. The G361 cells produce melanin for up to 50 population doublings. As the aim of this study is to compare the effect of blue light on the cell lines differing by morphology, transformation state, sensitivity to cell death, and origin, we decided to use the cell lines listed above. PC-3 cells are larger in comparison with small A2780 cells. Benign PNT1A cell line differs from malignant PC-3, and all four cell lines are derived from diverse tissues of origin. Furthermore, melanoma G361 cells expressing melanin may differ in the reaction of cells to blue light exposure.

Cell Cultivation

All four cell lines were cultivated in 25 cm² flasks with 5 ml of media at 37 °C in a humidified incubator (60%) with 5% CO₂ (Sanyo, Osaka City, Japan). Cell lines A2780, PNT1A and G361 were cultured in RPMI-1640 medium with phenol red indicator, L–glutamine, FBS and antibiotics penicillin/streptomycin (Sigma Aldrich Co., St. Louise, MO, USA). For the PC-3 cell line cultivation, Ham´s F-12 medium with FBS and antibiotics (Sigma Aldrich Co., St. Louis, MO, USA) was used. The same supplementation with antibiotics (penicillin 100 U/mL and streptomycin 0.1 mg/mL) and 10% FBS was used in both media. The cell medium was changed two times per week. Cell subculturing was done with 10% of trypsin solution (PAA, Pasching, Austria) with previous washing with EDTA (0.02% in PBS buffer).

QPI and Holographic Microscopy and Fluorescence Setting

QPI was performed by using a Q-PHASE multimodal holographic microscope (Telight, Brno, CZ). The Q-PHASE is equipped with fluorescence module using a halogen lamp as a non-coherent source of blue light. In this work, the module was used as a source of blue light for treatment of observed cell lines. The 485 nm light waves are emitted by the fluorescence light source of the attached module. Before the imaging experiment, cells were cultivated overnight in a concentration of 7000 cells/mL in flow chamber µ-Slide I Lauer Family (Ibidi, Martinsried, Germany). During the measurements, the chamber with cells was incubated in 37 °C humidified, 5% CO₂ atmosphere in H201–for Mad City Labs Z100/Z500 piezo Z-stages (Okolab, Ottaviano NA, Italy). Images and holograms were captured with lens Nikon Plan 10/0.3 and CCD camera (XIMEA MR4021 MC-VELETA, Münster, Germany) respectively. The fluorescence mode used was a plasma light source (Sutter Instrument Lambda XL Novato, CA, USA). Cells were irradiated with a 485 nm light with a 25 nm bandwidth. Light doses 0 mJ/cm², 24 mJ/cm² and 208 mJ/cm² were achieved by the combination of time exposition and light intensity.

The images were acquired automatically from seven positions every 3 min for 24 h. Holographic images were collected by custom software and raw data were numerically reconstructed. The numerical reconstruction was performed by custom software where the established methods of the fast Fourier-transform and phase unwrapping are implemented. The output from the software is an unwrapped phase image. This image has high intrinsic contrast and can be processed by an available image processing software. The unwrapped phase image is integrated phase shift through the cell and it is proportional to integrated cell dry mass density.