Pkh26 fluorescent cell linker pe conjugated dye

PKH26 fluorescent cell linker (PE-conjugated) dye was purchased from Sigma (USA) and used according to the manufacturer’s instructions [18 (link)]. OCCs were initially stained with PKH26 dye and subsequently resuspended in 3D media containing DMEM F-12 supplemented with 2% B27 (Invitrogen), 20 ng/mL VEGF (Peprotech), 20 ng/mL bFGF (Peprotech) and 5 ug/mL insulin (Sigma). The cells were plated at a ratio of 1/3 (eGFP + E4 + ECs/OCCs) at 60000/20000 cells per well of ultra-low attachment 24-well plates (Costar, Corning) and were grown in a humidified incubator at 37 °C with 5% CO₂. The media were changed every third day. Spheres were cultured for up to 5 days [15 (link)]. Fluorescence imaging was performed with an Evos^® FL digital inverted fluorescence microscope (AMG) or with a confocal microscope (see the confocal section).

PKH26 fluorescent cell linker (PE-conjugated) dye was purchased from Sigma (USA) and used according to the manufacturer's instruction. BCCs were initially stained with PKH26 dye and enriched as mammospheres in so-called 3D media under non-adherent condition in ultralow attachment plates (Corning, USA) following the method previously described by Dontu et al. [27] (link). The 3D media was made of DMEM-F12 (Sigma, USA) supplemented with 2% B27, 5 µg/mL insulin, 20 ng/mL basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). In order to prevent the formation of cellular aggregates, a highly viscose 3D media was prepared by the addition of 0.2% methylcellulose to the above mixture (Sigma, USA). To make mammospheres, PKH26⁺BCCs were seeded at 10³−5×10³ cells/mL of 3D media and cultured for 5–7 days to obtain primary mammospheres. Primary mammospheres were dissociated to single cells after 7 days by trypsinization and further sieving through 40- µm cell strainers and re-plated at 5×10³−10⁴ cells/mL to obtain secondary mammospheres. To form mammo-angiospheres, one part of PKH26⁺BCCs were mixed with 10 parts of GFP⁺E4-ECs (1∶10 ratio) and co-cultured under non-adherent condition for 5–7 days to obtain mammo-angiospheres. Sphere proliferation was measured by the increase in number of mammosphere clusters distinguished by PKH26 staining.