Celltiter 96 kit

The cytotoxicity of Fe₃O₄@C particles was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay using a CellTiter 96 kit (Promega, Madison, WI, USA) on A549 and AML12 cells. Cells were seeded at a density of 5×10³ cells per well in a 96-well plate with medium (100 μL) and incubated for 24 hours to allow them to adhere to the surface of the plate. Aliquots containing various concentrations of Fe₃O₄@C particles were added to each well, and the cells were incubated for an additional 48 hours. Prior to the addition of MTS reagent (20 μL) to the fresh culture media, Fe₃O₄@C solution was removed from the culture to prevent any interference in the absorbance measurement. Following incubation at 37°C for 3 hours, the absorbance was detected at 490 nm using a microplate reader (Multiskan; Thermo Scientific, Waltham, MA, USA). Cytotoxicity was expressed as the percentage of cell viability with respect to the control cells that had been treated with the vehicle of the particles only.

Cell media and reagents were purchased from Life Technologies (Carlsbad, CA, USA). All cell lines were obtained from ATCC (Manassas, VA, USA). Transfections were carried out using Neon nucleofection system from Life Technologies. Cell viability was assessed by trypan blue exclusion or by MTT cell proliferation assay using CellTiter 96 kit from Promega (Madison, WI, USA).

Solutol® HS 15 (BASF, Ludwigshafen, Germany) is a mixture of free polyethylene glycol 660 and polyethylene glycol 660 hydroxystearate. Labrafac® WL 1349 (Gattefossé Group, Saint-Priest, France) is a mixture of capric and caprylic acid triglycerides. NaCl was purchased from Prolabo (Fontenay-sous-Bois, France). Lipoïd® S75-3 (Lipoïd GmbH, Ludwigshafen, Germany) is a soybean lecithin made of 69% phosphatidylcholine, 10% phosphatidylethanolamine, and other phospholipids. Milli-Q water was obtained from a Milli-Q-plus® system (Merck Millipore, Billerica, MA, USA). Chitosan oligosaccharide lactate 5,000 Da with a degree of deacetylation ≥75% was purchased from Sigma Aldrich (St Louis, MO, USA). The dialysis membrane was purchased from Spectrum Laboratories (Rancho Dominguez, USA) and had a molecular weight cut-off point equal to 50,000 Da. O-Phthalaldehyde (OPA) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays (Cell Titer 96® kit) were purchased from Promega Corporation (Fitchburg, WI, USA).

Cell viability was assessed by MTS assay using a CellTiter 96 Kit (Promega, USA) according to the manufacturer's instructions. Trypan blue exclusion assay (C0011, Beyotime, China) were performed according to the manufacturer's instructions. The percentages of viable cells were measured by Countess II (Life technologies, USA). For FACS analyses, cells were grown to approximately 80% confluence in 6-well plates, washed with cold PBS and then fixed in 70% ethanol at 4°C overnight. The cells were subsequently stained with 50 μg/mL propidium iodide (PI) supplemented with 80 μg/mL RNase A at 37°C in dark for 1 h. The cells were then subjected to FACS analyses using a FACSCalibur Low Cytometer (Becton Dickson).