1 1 dioctadecyl 3 3 3 3 tetramethylindocarbocyanine perchlorate dii

The invasion assay was performed using Millicell™ hanging cell culture inserts (polyethylene terephthalate (PET) membranes with 8 μm pores) (Millipore). HMEC-1 cells (1 × 10⁵ cells per well) were plated on the upper chamber and allowed to grow to confluence, and then 10% Matrigel was loaded into the chamber. Tumor cells were treated with 50 ng/ml EGF in serum-free medium and then stained with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI) (Invitrogen) for 30 min. DiI-stained tumor cells (2 × 10⁵) were then loaded into the chamber, which was filled with serum-free medium, and incubated for 2 days. Cells on the apical side of each insert were scraped off. Invasion to the basolateral side of the membrane was visualized using an immunofluorescent microscope. The number of invading cells was determined in three randomly chosen fields under the microscope for three independent experiments.

The invasion assay was performed using Millicell™ hanging cell culture inserts (polyethylene terephthalate (PET) membranes with 8 μm pores) (Millipore). HMEC-1 cells (1 × 10⁵ cells per well) were plated on the upper chamber and allowed to grow to confluence, and then 10% Matrigel was loaded into the chamber. Tumor cells were treated with 50 ng/ml EGF or 10 μM PGE₂ in serum-free medium and then stained with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI) (Invitrogen) for 30 min. DiI-stained tumor cells (2 × 10⁵) were then loaded into the chamber, which was filled with serum-free medium, and incubated for 2 days. Cells on the apical side of each insert were scraped off. Invasion to the basolateral side of the membrane was visualized using an immunofluorescent microscope. The number of invading cells was determined in three randomly chosen fields under the microscope for three independent experiments.

Alexa Flour 647, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) was obtained from Invitrogen. Sephadex G-25 fine resin, egg phosphatidylcholine (PC), cholesteryl oleate, sodium cyanoborohydride (NaCNBH₃), triethylamine (TEA), 3-aminopropyl-triethoxysilan (APTES), ethanolamine (ETA), sodium deoxycholate and HEPES were from Sigma. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) was purchased from Avanti Polar Lipids. Cholesterol linked to boron dipyrromethene difluoride at sterol carbon-24 (cholesterol- Bodipy; C-Bodipy) was synthesized as described^{23 (link)}. Cholesteryl-ester- Bodipy (CE-Bodipy) was synthesized by conjugating linoleic acid to C-Bodipy as described²⁴ .

Cyclosporin A (CsA) was obtained from Pharma Stulln GmbH (Stulln, Germany). Kolliphor^® HS15 was obtained from BASF. Lipoid^® S75-3 was obtained from Lipoid GmbH (Ludwigshafen, Germany). Miglyol^® 812 (MCT) was purchased from Caesar & Loretz GmbH (Hilden, Germany). 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) was purchased from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N[maleimide(polyethyleneglycol)-2000] (ammonium salt) (DSPE-PEG2000-maleimide) was purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA) and cyclo(-Arg-Gly-Asp-D-Phe-Cys) acetate salt (RGD) from Bachem Distribution Service GmbH (Weil a. Rhein, Germany). Dulbecco’s phosphate-buffered saline (PBS) was acquired from Gibco^® Life Technologies (Waltham, USA). Purified water was obtained from a Milli-Q System from Millipore (Schwalbach, Germany). All other materials and reagents in analytical grade were obtained from Merck (Taufkirchen, Germany).

CHO-K1-hM₄R cells were grown as described above and seeded with a density of 25 000 cells per well into a µ-Plate 96 well Black plate (Ibidi, Gräfelfing, Germany) 5 h before the experiment. A stock solution of 1 mM 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) (Invitrogen, Eugene, Oregon, USA) in DMSO stored at −20°C was thawed and sonicated in an ultrasound bath for 5 min to disrupt aggregates. Cell medium was removed and replaced with 200 µl per well of 2 µM DiI in Dulbecco's phosphate-buffered saline (DPBS) with Mg²⁺ and Ca²⁺ (Sigma-Aldrich) to stain the cell membranes. The cells were incubated with the solution for 10 min before imaging. The cells were imaged with Cytation 5 cell imaging multi-mode plate reader equipped with 20X LUCPLFLN objective (Olympus) from Bright-field and RFP channels (LED light source with excitation filter 531(40) nm and emission filter 593(40) nm for RFP channel (BioTek Instruments) with the following parameters for bright-field: LED intensity = 4, integration time = 110 ms, camera gain = 24 and for RFP fluorescence channel: LED intensity = 1, integration time = 71 ms, camera gain = 24. The cells were imaged in the montage mode (196 locations) with Z-stack (10 planes, 4 planes below and 5 planes above focus) to cover any imaging location-dependent variability and simulate potential autofocusing errors.