In some experiments cell were fluorescently labeled with Red/Green/Blue cell tracker (Thermo Fisher, C34552, C7025, C2110 respectively). The stock solution was diluted 1 to 1000 in growth medium and cells were trypsinized and incubated in this medium for 30 min. Next, cells were washed twice with phosphate-buffered saline (PBS) (Lonza BE17-516F) to remove the excess cell tracker.
C2110
Manufactured by Thermo Fisher Scientific
The C2110 is a laboratory equipment item manufactured by Thermo Fisher Scientific. It serves as a core component in various scientific applications. The device's primary function is to perform specific tasks required in research and analysis settings, though the exact details of its intended use are not provided.
Automatically generated - may contain errors
Lab products found in correlation
C7025, by Thermo Fisher Scientific (1 mentions)
Be17 516f, by Lonza (1 mentions)
Model e800, by Hamamatsu Photonics (1 mentions)
Model c4742, by Hamamatsu Photonics (1 mentions)
Bz x710 fluorescence microscope, by Keyence (1 mentions)
Cc 4176, by Lonza (1 mentions)
Cls3471, by Corning (1 mentions)
Cc 3156, by Lonza (1 mentions)
3 protocols using c2110
1
Fluorescent Cell Labeling Protocol
2
Microscopy Analysis of Cellular Organelles
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For all microscopy experiments, cells were grown to mid-log, washed, and resuspended in SD-N for the time points indicated. Except for Figure 2B , deconvolved images were obtained using a Nikon microscope (Model E800) with a 100× objective using 1.2× camera magnification (Plan Fluor Oil, NA 1.3) and a CCD camera (Hamamatsu Model C4742). Data were collected using NIS software and processed using Image Pro software. All images of individual cells were optically sectioned (0.2-μm slices at 0.3 μm spacing) and deconvolved, and slices were collapsed to visualize the entire fluorescent signal within the cell. Linear quantification analysis was measured using the Image Pro software. The vacuoles were visualized in live cells either using mCherry-tagged Vph1, Pho8-BFP or staining with CMAC (Thermo, C2110; 100 μM) as described [21 (link)]. In Figure 2B a Keyence BZ-X710 fluorescence microscope with a 100× objective with 1.0× camera magnification (PlanApoλ Oil, NA 1.45) and a CCD camera was used. The images were deconvolved using BZ-X Analyzer software.
3
Multilineage Organoid Formation Conditions
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Fibroblasts, keratinocytes and endothelial cells were labelled with cell tracker fluorescent probes (C2110 excitation (ex) / emission (em) 353/466 nm, C7025 ex/em 492/517 nm, C34552 ex/em 577/602 nm; ThermoFisher) according to the producer's recommended protocol. The spheroid formation capacity of each cell type separately was evaluated by seeding 4 x 105 cells in 6-well ultra-low attachment culture plates (CLS3471; Corning) in the respective propagation media. For FSO formation, single-cell suspension consisting of 1.25 x 105 fibroblasts, 1.25 x 105 endothelial cells and 2.5 x 105 keratinocytes were prepared based on preliminary titration and seeded in various media conditions: (i) keratinocyte serum-free medium basal (17005075, Gibco), (ii) keratinocyte serum-free medium basal + supplements (EGF + BPE), (iii) keratinocyte serum-free medium basal + supplements + 10% hPL, (iv) endothelial cell basal medium (EBM, CC-3156, Lonza), (v) endothelial growth medium (EGM) = EBM + supplements (EGF, IGF, hFGF, VEGF, hydrocortisone, ascorbic acid; CC4176, Lonza), (vi) EGM incl. supplements + 10% hPL. Five days after cell seeding, areas of 1 mm² were counted to determine organoid numbers. Life cell imaging was performed for the first 48 hours; microscopic analysis was done 5 days after seeding (see image acquisition).
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