Trypan Blue

Cells were seeded on 35 mm cell imaging dishes (145 μm glass; Eppendorf, Enfield, CT, USA) at the seeding density required by each cell line (see table 2). Each plate was filled with 700 μl cell stock in the 18 mm cavity of the dish. The plates were left undisturbed in the incubator for 9–10 h to allow them to attach to the plate, except LNCaP cells, which had to be left undisturbed for 24 h. At this point, the plates were filled with 2 ml fresh media and returned to the incubator for 14 h (24 h for LNCaP) to complete the 24 h (48 h for LNCaP) before starting the experiment. The nanoparticle solution was prepared the day of the experiment (24 or 48 h after seeding). A volume of 2 ml was transferred from the nanoparticle stock (1.44 mg_Fe ml⁻¹) to a centrifuge tube containing 2 ml of fresh media (2X) and FBS, yielding a nanoparticle solution at 0.72 mg_Fe ml⁻¹. The order in which the plates were exposed to the magnetic field was obtained by a randomization tool. The process for each plate started by removing the culture media carefully at a specific region. Every pipetting step was done on the ‘S’ or south position of the plate. After media removal, the plate was filled with 500 μl of the nanoparticle solution or fresh media. After each run, the nanoparticle solution or the culture media was removed to wash the plate one time with buffer. Then, the buffer was discarded to fill the plate with 2.0 ml of fresh media. The dish was then moved to the incubator for 24 h or 48 h before detaching and suspending the cells to count them manually and assess cell viability with the Trypan Blue exclusion assay (see subsection C of the Supplementary Information) [23 (link), 58 (link), 59 (link)]. See figure 4 for a graphical depiction of the plate management. Each experiment consisted in three samples per condition and was repeated two other days for a total of nine samples per condition.
LIH: Experimental samples (E) were exposed to an AMF of approximately 42.6 kA m⁻¹ at 326 kHz for a maximum of 40 min. The treatment time was set to 30 min after reaching around 40.5 °C to maintain the temperature around 41 °C. This usually happened between 8–10 min. The AMF negative control (A) group was also subjected to the same conditions as the experimental sample. While these groups were being exposed to the AMF, two control groups were left in the incubator; one with only cells (C) and another group with cells and MNPs (N) for 40 min. This last group was used solely to ensure that the MNP concentration employed in the experimental group was not harmful (or contaminated) to the cells, at least, during the treatment time.
TRIH: Experimental samples (E) were exposed to an AMF of about 26.3 kA m⁻¹ at 303 kHz for a maximum of 40 min. The treatment time was set to 30 min after reaching around 41.5 °C in 5–8 min to maintain the temperature around 43 °C. Everything else was conducted in the same manner as described above.

The fluorinated oil was stored in a cell incubation at 37 ​°C and 5% ${\mathrm{C}\mathrm{O}}_2$ for overnight. Then the sorted and unsorted MCF-7 ​cells droplets were collected and sealed in a 1 ​ml tubes to prevent evaporation. Every 3 ​h, we transferred 10 ​μl of droplets to 200 ​μl tubes and recover cells in droplets by adding an emulsion breaker (Fluoro-Stop, Dolomite Microfluidics) in 1:1 ​vol ratio. The cell viability is determined by using trypan blue staining.