Co2 incubator

The current study was conducted using MCF-7 cells (ER-positive breast cancer cells), which were purchased from ATCC. The cells were grown in a CO₂ incubator (New Brunswick Scientific, Edison, NJ, USA) at 37 °C in using RPMI-1640 media, which was supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 g/mL). The samples were diluted to the desired concentration using serum-free media and treated for the required time period.

The rat heart-derived H9C2 cardiomyoblast cells were obtained from the National Centre for Cell Science (NCCS), Pune, India (originally from ATCC, USA). H9C2 cells were cultured with Dulbecco's modified Eagle's medium (DMEM) supplemented with penicillin (100 unit/ml), streptomycin (100 μg/ml), glucose (5.5 mM), L-glutamine (2 mmol/l), sodium bicarbonate (3.7 g/l), 10% fetal bovine serum (FBS), insulin (50 mg/ml), transferrin (27.5 mg/ml), selenium (0.025 mg/ml), and amphotericin B (5 μg/ml) in a humidified CO₂ incubator (New Brunswick Scientific, NJ, USA) with 5% CO₂ at 37°C [27 (link)].

About 100 μL of serial dilutions of single or combined extract or Dex were added to 1 × 10⁵ WBCs/well in 96-well plate and incubated in CO₂ incubator (New Brunswick Scientific, Netherlands) at 37 °C, 5% CO₂, and 90% relative humidity. After 72 h, 20 μL of MTT (5 mg/mL in PBS) was added to each well and incubated in CO₂ incubator for further 3 h, and then centrifuged for 10 min at 280 xg. One hundred microliters of DMSO was added (formazan crystals, MTT byproduct) and the absorbance was read at 570 nm using ELISA reader (BMG Lab Tech, Germany) [23 (link)]. Cell viability was determined and a relation between the cell viability and the studied extract or Dex concentrations was plotted for calculating the safe concentrations (EC₁₀₀, 100% cell viability).

The bone cells were isolated from the sterilized femur and tibia of three albino male rats (71–73 g) following the method of Wong et al.^{26 (link)} with some modifications. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Alexandria University and City for Scientific Research and Technology Applications (SRTA-City). All animal procedures were approved by the Animal Ethics Committee of the National Health and Medical Research Council policies and the Ministry of Health and Population, Egypt.
Bones were dissected into 1 mm pieces and washed well to remove blood, marrow cells, and any soft tissues. Bone pieces were placed in DMEM containing 100 U ml⁻¹ penicillin/streptomycin for 20 min in a CO₂ incubator (New Brunswick Scientific, Netherlands) at 37 °C, 5% CO₂ and 90% relative humidity. Bone cells were successfully isolated by digestion with 100 U ml⁻¹ collagenase type I and 25% trypsin. Cells were cultured with α-MEM containing 20% FBS in 6-well culture plate in a CO₂ incubator at the same previous conditions. After 24 h, the medium was aspirated and replaced daily thereafter until they reached confluence (90%) within 3 weeks. Then trypsinization was done for cells using trypsin (25%) containing EDTA (0.02%). After washing, cells were tested for the viability and counted using 0.5% trypan blue stain.

Two adherent human breast cancer cell lines MDA-MB-231 and MCF-7 and one suspension cell line of human malignant T-cells (HuT-78) were used to evaluate the cytotoxic effect of EA extract of P. oxalicum. HuT-78 cells were grown in 10% FBS containing Roswell Park Memorial Institute (RPMI) medium supplemented with 1% penicillin-streptomycin-amphotericin B solution (CELL clone) at 37 °C in a 5% CO₂ incubator (Eppendorf, Darmstadt, Germany). MDA-MB-231 and MCF-7 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (HI-FBS) (US grade, Thermo), and 1% penicillin-streptomycin-amphotericin B solution (CELL clone) and maintained at 37 °C in a 5% CO₂ incubator. The cells were observed at every 24 h for cell growth and the presence of any contaminants.

The cytotoxicity of the FFS of ZnO/CNC/PC III film was evaluated using the MMT (3-(4, 5 Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) colorimetric assay^{29 (link)}. Human colorectal adenocarcinoma cells (Caco-2, HTB-37) and normal lung fibroblasts (WI-38, CCL-75) obtained from the American Type Culture Collection (ATCC, Rockville, MD); were seeded independently in flat-bottom 96 well plate (Corning, USA) at a density of 1 × 10⁴ cell/well (100 µL) in high glucose DMEM culture medium (Biosera, France) supplemented with 10% fetal bovine serum albumin (Sigma-Aldrich, USA) and 1% Penicillin streptomycin (Sigma Aldrich, USA). Plates were incubated at 37 °C for 18 h in a humidified 5% CO₂ incubator (Eppendorf, Germany) to allow cells attachment. The FFS was serially two-fold diluted, added in triplicate in each well, and incubated for 48 h under the previously mentioned culture conditions. Supernatants from each well were withdrawn and replaced with fresh medium (100 µL/well) containing MTT reagent with a final concentration of 0.5 mg/mL, and plates were re-incubated at 37 °C for 4 h. Media over the cells were removed, and dimethyl sulfoxide (Merck, Germany) was added (100 µL/well), plates were shaken for 15 min, and the absorbance was measured at 570 nm using a microplate reader (AccuReader M965 + , Metertech, Taiwan).

All cultures were performed at 37 °C in a 5% CO₂ incubator (Eppendorf). For more details, see Supplemental Methods.