Ix 71s8f 3

For light microscopy, the cells (1×10⁵ at time point 0 h) were cultivated directly on glass microscopy slides (75×25 mm, thickness 1 mm) in medium without and with 1 μM sarcosine. Prior microscopic examination, slides with a monolayer of cells were removed from Petri dishes, rinsed with a medium and PBS and directly used for investigation of their density under an inverted microscope (Olympus IX 71S8F-3, Olympus, Tokyo, Japan).

The tumor, heart, kidneys, and liver of each mouse were investigated to assess tissue microstructure, DOX distribution and iron distribution. The samples were fixed in formaldehyde (10%, v/v) overnight, subsequently dehydrated in a series of progressively more concentrated ethanol and embedded in paraffin wax. Sections were cut at 5 µm, mounted on glass slides, deparaffinized and either subjected to fluorescent detection of DOX distribution or stained with hematoxylin-eosin for assessment of structure and Perls’ Prussian blue for detection of iron. The microscopical observations were conducted using Olympus IX 71S8F-3 (Olympus, Tokyo, Japan).
Quantitative DOX detection was performed via the acidified isopropanol extraction according to a previously reported procedure^{20 (link)}. Briefly, approx. 0.02 g of tumor, heart, liver and kidney collected from all groups was homogenized with an addition of 1:10 (wt/vol) acidified (0.75 M HCl) isopropanol on ice and extracted overnight at −20 °C in the dark. The samples were then centrifuged at 4 °C and 18000 g for 10 min. DOX fluorescence (excitation at 480 nm, emission at 600 nm) was measured in the supernatant with correction of tissue autofluorescence using the homogenates from saline-injected controls. The results were presented as µg per gram of tissue and expressed as means ± SD for a group ( $n=3$ mice per group).

The samples were fixed in formaldehyde (10%) overnight, subsequently dehydrated in serial ethanol concentrations and embedded in paraffin wax. Sections were cut at 5 μm, mounted on glass slides, deparaffinized and stained with hematoxylin-eosin. The microscopic observations were conducted using an Olympus IX 71S8F-3 (Olympus, Tokyo, Japan).