Cell imaging coverglass

To determine the rate of Dox binding to the nucleus the following in vitro experiments were performed. GL261 cells were seeded at a density of 20,000 cells/ml in cell imaging slides (Cell imaging coverglass, Eppendorf). At 60-70% confluency, free-Dox solution of 10 μM was applied to the imaging slide and fluorescence intensity was measured using confocal microscopy (LSM 710, Zeiss) every 5 min for 1 h. This concentration was selected because it produced images with high signal to noise ratio (SNR). In these measurements, the following 3 protocols were tested: 1) 37 °C, 2) Heat at 41.5 °C for 1 h, and 3) Heat at 41.5 °C (10min) + 37 °C (during imaging). The latter was a similar procedure as the one followed for the mouse studies.

Purified BM CD115⁻ and CD115⁺ M-MDSC from EL4 or LLC1 TB mice were cultured in a 12-well plate at a density of 1.5 × 10⁵ cells/well. RPMI complete medium supplemented with 1 mM sodium pyruvate (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 55 μM 2-Mercaptoethanol (Gibco), and 10 ng/ml GM-CSF (PeproTech, Rocky Hill, NJ, USA) was used as a base medium. To mimic the tumor microenvironment, EL4 or LLC1 tumor explant supernatants (TES) were prepared according to the previous study^{67 (link)}. Briefly, one gram of EL4 or LLC1 tumor was minced and cultured in RPMI complete medium for 24 h and supernatant was collected and stored at −80 ^oC until use. For M-MDSC differentiation, 5% EL4 or LLC1 TES was added to the base medium. On day 3, the supernatant from TES free wells was collected as a condition media (CM) for migration and invasion assays. After collecting supernatant, each well was refilled with new media and cultured for an additional three days. Cells were collected for flow cytometric analysis after 6 days of culture. Some cells were separately cultured in Cell Imaging Coverglass (Eppendorf, Hamburg, Germany) and stained with hematoxylin-eosin (H&E, Merck Millipore, Burlington, MA, USA) for microscopic imaging.

MCF-7 cells were plated in 6-well plates before harvesting. After treatment and washing, the cells were fixed with 4% paraformal-dehyde solution, followed by washing and blocking. Next, the cells were incubated with the FITC-conjugated anti-P-gp polyclonal antibody or an isotype-matched negative control for 1 h at 37 °C (BD Biosciences, Franklin Lakes, NJ, USA). After washing, the cells were analysed via flow cytometry and the data were acquired on a BD FACSVerse (FACSCalibur, BD, Franklin Lakes, NJ, USA) and then were analysed with CELLQUEST software. MCF-7 cells were plated in Cell Imaging Coverglass (Eppendorf, USA), after treatment and washing, incubated with the FITC-conjugated anti-P-gp polyclonal antibody overnight at 4 °C and then laser confocal scanning microscopy analysis was performed (Live 5, Carl Zeiss, Oberkochen, Germany).