Transwell inserts (6.5 mm diameter, 8 μm pore size; Corning) were coated on the top with 100 μl growth factor–reduced Matrigel (356231; BD Biosciences). 5 × 104 cells were seeded on the bottom of the Transwell inserts and allowed to adhere for 4 h. Transwell inserts were inverted, and the medium containing 10% FCS and TGFβ/EGF was added to the top as the chemoattractant, and in the lower compartment, the medium containing just 0.5% FCS was added. Cells were incubated for 3 d and then stained with 10 μM CellTracker CMFDA dye (C2925; Thermo Fisher Scientific) for 1 h at 37°C. Invaded cells that crossed the membrane pore and invaded the Matrigel were imaged by confocal microscopy (Zeiss LSM 880) with a 20x objective with optical Z-sections scanned at 2-μm intervals moving up from the underside of the membrane into the Matrigel. The results were quantified from three randomly chosen views from each insert.
Celltracker cmfda dye
Manufactured by Thermo Fisher Scientific
CellTracker CMFDA dye is a fluorescent probe designed for cell labeling and tracking in live-cell imaging applications. It is a cell-permeant dye that becomes fluorescent upon internalization and can be used to visualize and monitor cell populations over time.
Automatically generated - may contain errors
Lab products found in correlation
Ix70 inverted microscope, by Olympus (2 mentions)
Lsm 880, by Zeiss (1 mentions)
Matrigel, by BD (1 mentions)
Transwell insert, by Corning (1 mentions)
Huvecs, by Lonza (1 mentions)
Dynabeads epithelial enrich, by Thermo Fisher Scientific (1 mentions)
Imipramine, by Merck Group (1 mentions)
Epcam microbeads, by Miltenyi Biotec (1 mentions)
Fluorescence activated cell sorting (facs), by BD (1 mentions)
Dead cell removal microbeads, by Miltenyi Biotec (1 mentions)
6 protocols using celltracker cmfda dye
1
Transwell Cell Invasion Assay
2
Quantifying Adherent Cell Migration
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Flat glass-bottom 96-well plates were precoated with fibronectin (as in the 2D migration assay). 1 × 106 cells/ml were labeled with 1 μM CellTracker CMFDA dye (C2925; Thermo Fisher Scientific) for 40 min at 37°C. The labeled cells were washed and resuspended in HBS solution (0.14 M NaCl, 5 mM KCl, 1 mM CaCl2, 0.4 mM MgSO4, 0.5 mM MgCl2, 0.3 mM Na2HPO4, 6 mM glucose, 4 mM NaHCO3), and 3 × 104 cells in 100 μl were added in each well of the 96-well fibronectin-coated plate. Plates were incubated for 30 min at 37°C to let the cells adhere and then carefully washed twice with PBS to remove the non-adherent cells. Adherent cells were fixed in 100% methanol for 10 min and visualized with an Olympus IX 70 inverted microscope at 10x magnification. Samples were analyzed in triplicates, and three fields of view were imaged per well. Cells per field of view were quantified using Fiji software.
3
Quantifying Lung Cell Adhesion via Microscopy
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Mouse lungs were placed in OCT compound (Tissue-Tek 4583), snap-frozen, and kept at −80°C until the experiment. Lung cryosections 8 μm thick were placed on glass slides. A chamber was created by gluing the neck, cut from a 1.5-ml micro-centrifuge tube, around the lung section. Cells were labeled with CellTracker CMFDA dye (C2925; Thermo Fisher Scientific), for 40 min at 37°C, washed, and resuspended in HBS solution, and 0.25 × 106 cells in 500 μl were added to each chamber. Glass slides with cells were placed on a shaker and slowly agitated for 30 min at room temperature. Non-adherent cells were removed by washing four times with PBS. Adherent cells were fixed in 100% methanol for 10 min and imaged with an Olympus IX 70 inverted microscope at 10x magnification, and two to three fields of view were imaged per lung section. Each sample was analyzed in duplicates. Fiji software was used to quantify the number of cells and the lung area per field of view. The average number of cells per unit lung area was calculated and normalized to the control sample.
4
Wound Healing Assay with IgG Fc Variants
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HUVECs (Lonza) treated with cell-tracker CMFDA dye (Thermo Fisher) were seeded in 24-well plates coated with rat tail type I collagen (354,236; BD Biosciences, Franklin Lakes, NJ) and “scratch-wounded” using a 200 μl pipette tip. After wounding, cells were treated with different concentrations (5, 10 ug/ul) of either IgG Fc, N110–14Fc, or N410–14Fc. After approximately 14 h, microscopy was used to image cell migration to the scratch.
5
Gene Expression Analysis of Co-cultured Cells
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To be able to differentially assess gene expression of both cell types after co-culture, spheroids were dissociated using Cell Passaging Solution (Accutase; Pelo Biotech, Martinsried, Germany). After achieving a single cell suspension, fractions of ASCs and breast cancer cells were sorted utilizing Dynabeads®Epithelial Enrich (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Subsequently, mRNA was isolated from both cell fractions as described for qRT-PCR. To exclude the impact of dissociation and sorting procedure on gene expression, respective controls were kept and analyzed as well. The dissociation and sorting procedure per se had no influence on the gene expression of target genes. For the evaluation of separation efficacy, ASCs were pre-stained with CellTracker™ CMFDA dye (Thermo Fisher) and the proportion of each cell type was counted in both fractions.
6
Cytotoxicity Assay for Tumor-Specific CTLs
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To assess the cytotoxicity of ESO CTLs, MCF-7 breast cancer cells were transduced with NY-ESO-1 and CLEC2D lentiviral vectors (NY-ESO-1+ CLEC2D+ MCF-7). NY-ESO-1+CLEC2D+ MCF-7 cells were labeled by CellTracker CMFDA Dye (Thermo Fisher Scientific, #C7025) and passed through Dead Cell Removal Microbeads (Miltenyi Biotec, #130-090-101), then cocultured with ESO CTLs at an effector/target ratio of 1:1 for 8 hours. For the cytotoxicity of TILs, primary breast cancer cells were isolated from tumor tissue and purified by EpCAM Microbeads (Miltenyi Biotec, #130-061-101). Then celltracker-labeled primary tumor cells were passed through Dead Cell Removal Microbeads and cocultured with autologous pentamer+ CTLs (CTLs isolated by FACS using MUC1 pentamer) at an effector/target ratio of 1:1 and autologous CD161+ CTLs at an effector/target ratio of 10:1 for 12 hours. In some experiments, CD161+ and CD161– T cells were separated and seeded into 96-well plates via FACS (BD Influx). At the end of the coculture, all cells were stained with PI (eBioscience, # 00-6990, 1:50) and analyzed by flow cytometry immediately. In some experiments, T cells were preincubated with imipramine (Sigma-Aldrich, #I0899, 100 μmol/L) for 1 hour before coculturing with autologous tumor cells.
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