AMF treatments of MNPs and MNP-loaded cells were performed at 37°C in airtight conditions. The temperature of cell pellet was recorded by the infrared thermometer (OS 3708; Omega Engineering,
Stamford, CT, USA). Cell viability assay: MTT assay and trypan blue assay MTT assay Cell viability was measured using 3-(4,PHA-848125 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich Company selleck chemicals llc Ltd., Gillingham, Dorset, UK) assay. After being treated in AMF, HeLa cells were reseeded into 96-well petriplate for 2 h incubation in quintuplicate. Following incubation, 20 μL MTT (5 mg/mL in PBS) solution was added to each well and incubated for another 4 h. After that, the culture supernatant was extracted, and purple insoluble MTT product was re-dissolved in 150 μL dimethyl sulfoxide. Lastly, the concentration of the reduced MTT in each well was measured at 570 nm using a microplate
reader. It is notable that the untreated MNP-loaded cells (i.e., the 0 min group) were used as control and absorbance OICR-9429 nmr was adjusted by correcting for the bias caused by the dark MNPs. Trypan blue assay After being treated with AMF, the medium was removed and the cells were stained by 0.4% trypan blue (Sigma-Aldrich Company Ltd., Gillingham, Dorset, UK) solution for 3 min. The cells with damaged cell membranes were stained by trypan blue and counted under the optical microscope. The above tests were repeated three times. Optical images of cellular semi-thin sections, SEM of cell surface, and TEM of cellular ultramicrocuts The HeLa cells were firstly fixed by adding 0.5% and 2% (w/v) glutaraldehyde and kept for 1 h Cell Penetrating Peptide at room temperature. Then the cells were dehydrated with ethanol in
series of concentrations 50%, 70%, 80%, 90%, and 100% (v/v) for 10 min respectively. Finally, the acetone-infiltrated cells were embedded in resin, and the blocks containing the cells were cut into thin sections in 500 or 50 nm using a diamond knife. For TEM of internal cell structure, the 50-nm ultramicrocuts were transferred into a copper grid for viewing. For optical macroscope viewing (6XB-PC, Shanghai Optical Instrument Factory, Shanghai, China), the 500-nm semi-thin sections were observed. For scanning electron microscope (SEM; LEO1530VP; LEO Elektronenmikroskopie GmbH, Oberkochen, Germany) of cell surfaces, the dehydrated cells were conductively coated and observed at 5 kV. Results and discussion Materials characterization TEM images of MNPs (Figure 2) revealed that most spherical MNPs were of a diameter of 200 ± 50 nm, while minority of MNPs was smaller. For rod-shaped MNPs, length was 200 ± 50 nm and diameters ranged from 50 to 120 nm. XRD patterns revealed that both types of MNPs were pure Fe3O4 (JCPDS no 19-0629). Meanwhile, the relatively strong (311) peak of rod-shaped MNPs implied that the crystals grow along the (311) crystallization plane to form rods. The saturation magnetic inductions for the MNPs were similar: 70.