Pkh26 fluorescent cell linker

Human blood PLTs and hESC‐derived PLTs were resuspended in modified Tyrode's buffer and labelled with a PKH26 Fluorescent Cell Linker (10 µmol/L, Sigma‐Aldrich). Human blood PLTs (labelled with PKH67, 6 × 10⁷) were mixed with fluorescence‐labelled human blood PLTs (3 × 10⁶) or hESC‐PLTs (3 × 10⁶), treated with thrombin (5 U/mL), and stirred at 1200 rpm at 37°C to trigger PLT aggregation. PLT microaggregates in 50 µL buffer were spread onto glass slides and visualized under a fluorescence microscope (GE Healthcare).

For CLSM analysis, SW620 and CTSC#18 cells were stained with PKH26 Fluorescent Cell Linker (Sigma-Aldrich), seeded and treated 24 h later with combined drugs for 48 h. Then, untreated and treated CRC cells were co-cultured with IFN-α-DCs for 4 h. Cells were fixed and images were observed through a 20X objective lens. Images were taken on an inverted microscope equipped with a confocal spectral imaging system (Olympus Fluoview 1000). Excitation light was obtained by a Diode Laser HeNe (561 nm) for PE. PE-Red emission was recorded from 583 to 628 nm. Images were analyzed by the C1-LCSI EZ-C1 Software.

IFN-DCs were generated using a standard protocol previously described^{59 (link)}. Briefly, CD14⁺ monocytes were isolated from human peripheral blood mononuclear cells (PBMCs) and cultured with both 500 U/ml GM-CSF (PeproTech, NJ) and 10⁴ IU/ml IFN-α2b (Intron A, Merck Sharp and Dohme, Kenilworth, NJ) for 3 days. Then, the floating cells were collected and used for experiments. CD14⁺ monocytes were also cultured for 3 days with GM-CSF alone, obtaining GM-DCs that were used as control in the quantitative real-time PCR (qPCR) experiments. For 3D microfluidic migration experiments, IFN-DCs were stained with PKH-67 Fluorescent Cell Linker (Sigma-Aldrich), according to the manufacturer’s instructions. Low passage-number SW620 CRC cells were maintained in RPMI-1640 (Lonza, Verviers, Belgium) with 10% FCS (EuroClone, West York, UK). Cells were seeded at 3.5 × 10⁴/cm² and then 24 hour later were treated with or without 2 nM romidepsin® and 10⁴ IU/ml IFN-α2b (I) in combination (RI) for 48 h. For 3D microfluidic migration experiments, SW620 cells were stained with PKH-26 Fluorescent Cell Linker (Sigma-Aldrich), according to the manufacturer’s instructions.