MCF7 control or JNK1/2 knockdown cells were labeled with 10 μM Cell Tracker dyes (green or red; Life Technologies) for 30 min at 37°C and then plated at a 1:1 ratio at a total cell density of 400,000 cells per 35-mm glass-bottom dish, in the presence of Y27632 to block entosis during plating overnight. Y27632 was then washed out three times with PBS the next day and the cells were exposed to UV radiation. 24 h later, the cells were analyzed by confocal microscopy as described above and previously (Hamann et al., 2017 (link)), where heterotypic cell-in-cell structures were counted and the number green-in-red and red-in-green structures were determined.
Cell tracker dyes
Manufactured by Thermo Fisher Scientific
Cell Tracker dyes are fluorescent probes designed for labeling and tracking live cells. They passively diffuse into cells and become fluorescently labeled, enabling the visualization and monitoring of cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Vybrant dye, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Y 27632, by Bio-Techne (2 mentions)
Evos fl auto, by Thermo Fisher Scientific (1 mentions)
Red cmptx, by Thermo Fisher Scientific (1 mentions)
Tissue culture inserts, by Corning (1 mentions)
Rat tail collagen 1, by Corning (1 mentions)
Fluoro gel with tris buffer, by Electron Microscopy Sciences (1 mentions)
Photomultiplier tube, by Hamamatsu Photonics (1 mentions)
Fluoromount g, by Electron Microscopy Sciences (1 mentions)
Fv1200, by Olympus (1 mentions)
Fv1000, by Olympus (1 mentions)
Fluoview software, by Olympus (1 mentions)
7 protocols using cell tracker dyes
1
Quantifying Heterotypic Cell Internalization
2
Optimizing Cellular Dye Labeling Protocols
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To test cellular dyes, the cells are plated on collagen-coated tissue culture plastic, fluorescently labeled with a range of concentrations of various CellTracker dyes (Life technologies) and Vybrant dyes (Life Technologies) according to the manufacturer’s suggested protocol and maintained in respective serum-free media. Cells are also tested in varying media compositions to determine optimal viability. Tested media compositions include basal astrocyte medium alone (Sciencell) or supplemented with 1% B27 without vitamin A and 0.5% N2 and/or 0.01% FGF and 0.1% EGF. For all tests, the growth of labeled cells is measured after 18, 48, and/or 72 h using the CCK-8 cell proliferation and cytotoxicity (Dojindo) kits according to manufacturers’ suggested protocols. After 72 h, cells are also assessed for viability using Live and Dead ReadyProbes Reagents (Life technologies), imaged using wide-field microscopy with EVOS FL Auto (Life technologies), and quantified using ImageJ (National Institutes of Health). Each CellTracker or Vybrant dye test was performed similarly for each glial cell type (Supplementary Fig. 3 ).
3
Imaging Entosis in MCF-7 Cells
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MCF-7 cells (Lombardi Cancer Center, Georgetown University, Washington, D.C.) were cultured in DMEM (11965-092; Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (F2442; Sigma-Aldrich) and penicillin/streptomycin (30-002-CI; Mediatech). Cells expressing the H2B–mCherry nuclear marker were prepared by transducing cells with retroviruses made with the pBabe–H2B–mCherry construct, as described (Florey et al., 2011 (link)). Cells were transfected with expression constructs by nucleofection (Cell Line Nucleofector Kit V, VCA-1003; Lonza, Basel, Switzerland) according to manufacturer’s protocol and assayed 24 hours post transfection. The following expression constructs were used: pQCXIP–GFP and GFP–C1–PLCδ-PH (a gift from Tobias Meyer (Addgene plasmid # 21179; http://addgene.org/21179 ; RRID:Addgene_21179) (Stauffer et al., 1998 (link))). To observe tail formation and cleavage during entosis, MCF-7 cells were labeled with 10 μm Cell Tracker dyes (green or red, C7025 and C34552, respectively; Life Technologies, Grand Island, NY) for 20 minutes at 37°C, then plated at a 1:1 ratio at a total cell density of 250,000 cells in 35 mm glass-bottom dishes overnight in media containing 10 μm Y-27632 (#1254, Tocris Bioscience) to block entosis. The next day cells were washed three times with PBS, fresh media was added, and analyzed by confocal microscopy.
4
Fluorescent Cell Staining for Imaging
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CellTracker™ dyes (Life-Technologies), Red CMPTX (RC) and Deep Red (DR), were used to fluorescently stain cells. Prior to printing, the cells were suspended in serum-free culture medium containing either 5 μM RC or 1 μM DR (Supplementary Methods). The cells stained were: HEK-293T, CFP expressing HEK-293, primary chondrocytes and oMSC-derived osteoblasts. Printed constructs were imaged by fluorescence confocal microscopy (Leica SP5) and wide-field light microscopy (Leica DMI 8).
5
In Vitro 3D Brain Tumor Model
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Experiments are carried out in tissue culture inserts with 8 µm pore size (Sigma Aldrich CLS3374). Cells are fluorescently labeled with CellTracker dyes (Life technologies) and Vybrant dyes (Life Technologies) according to the manufacturer's suggested protocol. GBM cells (5.0 × 105), astrocytes (8.0 × 104), and microglia (8.0 × 104) are seeded in 75 µL gel comprising (0.2% hyaluronan; ESI Bio) and 0.12% rat tail collagen I (Corning) according to cell ratios quantified from human sections. The gels are plated within 96-well tissue culture inserts with 8 µm pores (Corning) and cross-linked at 37 °C in a humidified incubator containing 5% CO2 and 21% O2. After 3 h, serum-free medium (Astrocyte Basal; Sciencell, with 1% B27, 0.5% N2) is added to the top and bottom of each tissue culture insert. For static conditions, the medium level is consistent inside and outside the insert, while the medium on top of the gel is greater in flow conditions. This equates to 25 µL of medium under and 125 µL of medium on top for flow, and the reverse in static. Hydrogel stiffness was previously optimized to recreate mechanical properties of the brain22 (link).
6
Synchronized Lamellipodia Imaging Protocol
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Cells were plated on coverslips coated with 10 µg/ml fibronectin and left to spread overnight before either being fixed with 4°C 4% PFA in Krebs-S buffer (for unsynchronized populations) or treated to synchronize lamellipodia. Lamellipodial synchronization was achieved by addition of 150 µM CK-666 for 2 h, followed by washout of the drug with regular DMEM for 10 min (unless otherwise specified) before fixation. Cells were then permeabilized in 0.1% Triton X-100 for 5 min. 5% BSA/NGS mixture was used to block for 1 h. Primary antibody was added either at 4°C overnight or for 2 h at room temperature. Secondary antibody was added for 1.5 h at room temperature. Coverslips were washed thoroughly with PBS and then mounted using Fluoromount-G (Electron Microscopy Sciences) or Fluorogel with Tris buffer (Electron Microscopy Sciences). In comparative experiments, control and test cells were plated in mixed populations on the same coverslip, and GFP expression or Cell Tracker dyes (Life Technologies) were used to identify populations. Coverslips were imaged on a confocal microscope (FV1000 or FV1200; Olympus) with a photomultiplier tube (Hamamatsu Photonics) controlled by Fluoview software (Olympus) with a 40× 1.3 NA objective lens (Olympus) at room temperature. Micrographs are displayed as maximum intensity projections of z stacks.
7
Imaging Entosis in MCF-7 Cells
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