2.7. Culture and Treatment of Cancer Cell Line

Human NSCLC cell lines, epithelial lung carcinoma, and A549 obtained from ATCC, Manassas, VA, USA, were grown in non-coated T75 culture flasks as shown in Figure 1. Cells were filled with a complete medium: Dulbecco’s modified eagle medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (PS) in a humidified 5% CO₂ atmosphere at 37 °C. The medium was changed every third day and the cells were harvested and separated using 0.05% trypsin when they have grown to subconfluence. They were transferred to a 96-well plate (5 × 10⁴) cells/well to study the cytotoxicity of E. camaldulensis and CDDP on A549 cells.

A549 cells captured at the American University of Beirut (AUB Lab).

MCF-7 cell line obtained from ATCC, Manassas, VA, USA, is a widely studied epithelial cancer cell line derived from breast adenocarcinoma, has characteristics of differentiated mammary epithelium. These cells grow adherently as a monolayer, and were cultured in the same manner as A549.

Human peripheral blood mononuclear cells (PBMCs) obtained from ATCC, Manassas, VA, USA, comprising lymphocytes (B-cells, T-cells, and NK-cells), monocytes, and dendritic cells, were frequently used for the evaluation of immune responses.

For the ethanolic extract, the powder was dissolved using vortex in the DMSO solvent, such that the DMSO concentration does not exceed 0.5% or else it will be toxic to the cells, while for the aqueous extract, the powder was dissolved using vortex in Dulbecco’s modified eagle medium (DMEM). The two extracts were filtered by a filter of diameter of 0.2 μm. The prepared concentrations for both ethanolic and aqueous extracts were: 75, 150, 300, 600, and 900 μg/mL.

For CDDP, the tested concentrations were prepared from a stock solution (1000 μg/mL), the obtained treatment test concentrations were: 0.5, 1, 2, 4, 8, and 12 μg/mL.

Combinations of E. camaldulensis extracts and CDDP were also prepared for each extract.

A total of 70% to 80% confluent cells were seeded in a 24-well tissue culture microplate at the density of 100 × 103 cells/well and then treated with the prepared concentrations of E. camaldulensis and CDDP for 24 h. Evaluation of the anticancer activity was performed by measuring the cell viability of the A549, MCF-7, and PBMC by the neutral red assay after transferring the cells into a 96-well plate. Or, cells were directly seeded in a 96-well plate at a density of 10 × 10³ cells/well and then treated with the prepared concentrations of E. camaldulensis and CDDP for 24 h; where cell viability of the A549 was assessed by the MTT Assay.

Fifteen milliliters of blood were collected from a donor, and then 15 mL of PBS were added to them with a proportion (1:1). Fifteen milliliters of ficoll were added to the 30 mL of the prepared diluted blood, with a proportion (1:2). Centrifugation was done for 30 min at 400 RCF, where T (temperature) was 20 °C. The PBMC layer was removed and taken as a buffy white coat. PBMC was washed by 10 mL PBS. Centrifugation was done again for 15 min at 200 RCF to remove PBS. Another wash was done by 15 mL PBS to further purify the cells. Centrifugation was done again for 15 min at 200 RCF to remove PBS. The pellet containing the PBMC was taken, where the cells were resuspended in RPMI. PBMC was counted and finally seeded in wells. Finally, PBMC was subjected to treatment with E. camaldulensis and CDDP in the same way as A549, where the viability of cells was tested by the neutral red assay.