br The chemical composition of simulated body fluid is presented

The chemical composition of simulated body fluid is presented in Table 1.
2.4. Hyperthermia effect
To evaluate the heat generation of synthesized magnetic HT powder and as-received magnetite powder, magnetic powder with the con-centration of 50 mg/mL was dispersed in distilled water. 2 mg/mL so-dium pyrophosphate was added to solution as a surfactant to decrease agglomeration effect of nano-sized particles. Dispersed powder samples
Table 1
Chemical composition of simulated body fluid.
No. Reagent used Amount in 1000 mL distilled water
were exposed to AMF with 45.2 G field strength by using a coil of an inductive heater with 3 cm diameter (Power cube 60 Hz, 110v; AREZZO). Additionally, temperature was recorded by a thermometer (Luxtron One, Lambda Photometrics).”
2.5. Cell culture and powder seeding
Two types of CFTRinh-172 including human mesenchymal stem cells (hMSCs) derived from human bone marrow (Lonza, Allendale, NJ) and tumor osteosarcoma cell line Saos-2 (ATCC HTB-85, ATCC, Manassas, VA, USA) were used to evaluate biocompatibility and hyperthermia abilities of magnetite and synthesized 0.2Fe-HT powders. The cells were cultured in Dulbecco Modified Eagle's Medium (DMEM, Corning), containing 1% penicillin/streptomycin (Invitrogen) and 10% fetal bo-vine serum (FBS, Atlanta Biologicals). They were maintained in a hu-midified incubator at 37 °C with 5% CO2 and passaged at ~80% con-fluency. On the day of the experiment, powder samples were autoclaved and an equal-weight of each sample (~50 mg) was placed at the bottom of 48-well tissue culture plates. Each well was seeded with 15,000 cells and incubated for up to 4 h. After that, the media was replaced with fresh media before putting the samples back in the in-cubator. Media was replaced every 3 days during the experiment.
To study in vitro hyperthermia efficiency of magnetite and synthe-sized 0.2Fe-HT powders, plates containing samples and human cervical cancer Saos-2 cells were exposed to AMF with 45.2 G field strength by using a coil of an inductive heater with 3 cm diameter (Power cube 60 Hz, 110v; AREZZO). Temperature fluctuation in the wells was measured by a thermometer (Luxtron One, Lambda Photometrics). All the samples were maintained at the temperature in the range of 42–44 °C by using a temperature sensor (NTC thermistors, Germany). We controlled the temperature in that range by designing a sensor worked with AMF. The alternating magnetic field was turned on and off when the temperature of wells went below 42 °C and above 44 °C, re-spectively. External alternating magnetic field was applied during whole experiment period (7 days). Both external magnetic field and remanent magnetization of particles are the main agents of heat gen-eration. All sensors and thermometers were perfectly sterilized. The thermometers were at the top of wells, soaked in media and without any direct contact with cells.
The metabolic activity of cells was assessed on days 1, 3, and 7 of culture using PrestoBlue assay (Invitrogen). As per the manufacturer recommendation, the assay reagent was diluted 1:10 in culture media and incubated with samples for 1 h. The fluorescent intensity, which is an index for metabolic activity, was measured using a BioTek Synergy 2 plate reader. In addition, the viability of the cultures was assessed employing a Live/Dead assay kit (Invitrogen, USA), where staining of live or dead cells is done by calcein-AM (ethidium homodimer-1). The live/dead solutions were diluted in phosphate buffered saline (PBS) at a ratio of 1:2000 and 1:500, respectively. The samples were incubated for 20 min at 37 °C and visualized for fluorescence signals using a fluor-escent microscope (Zeiss) where the live and dead cells would appear in green and red color, respectively.