Non-small cell lung cancer cell line (H1650) cells were fluorescently-labeled with green cell tracker dye (Invitrogen). 10,100 or 1,000 labeled cells were spiked into whole blood drawn from healthy donors and flowed through the CTC capture device using a syringe pump. The number of cells captured was enumerated and capture efficiency was calculated by dividing this number by the initial cell number prior spiking. Cells in the device were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) nucleus dye (Invitrogen). All experiments were conducted in triplicate.
Celltracker green dye
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
CellTracker Green dye is a fluorescent stain used for labeling live cells. It can be used to track and visualize individual cells in a population. The dye passively diffuses into cells and becomes fluorescent upon reaction with intracellular components.
Automatically generated - may contain errors
Lab products found in correlation
Celltracker red dye, by Thermo Fisher Scientific (6 mentions)
μ slide chamber, by Ibidi (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Jsm 7000f sem, by JEOL (1 mentions)
Dcp 1, by Denton (1 mentions)
Sytox blue nucleic acid stain, by Thermo Fisher Scientific (1 mentions)
Te300, by Nikon (1 mentions)
Axiovert 200m inverted microscope, by Zeiss (1 mentions)
Live cell observer, by Zeiss (1 mentions)
Axiocam mrm camera, by Zeiss (1 mentions)
Rat tail collagen type 1, by BD (1 mentions)
Incucyte flr, by Sartorius (1 mentions)
Facsaria 2, by BD (1 mentions)
Cytodex 3 beads, by Merck Group (1 mentions)
Fgf 2, by Lonza (1 mentions)
Lsm 780 confocal scanning microscope, by Zeiss (1 mentions)
Vegf a, by Lonza (1 mentions)
D erythro sphingosine 1 phosphate, by Avanti Polar Lipids (1 mentions)
Egm 2mv medium, by Lonza (1 mentions)
Confocal laser scanning microscope, by Zeiss (1 mentions)
21 protocols using celltracker green dye
1
CTC Capture Device Efficiency Analysis
2
Quantifying Cytotoxicity of Labeled T Cells
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On the basis of
our previous studies, a ratio of 100:1 for effector (p98 T cells)
to target (MMC cells) was selected for this study.25 (link),26 For fluorescence imaging, p98 T cells were labeled with Green Cell
Tracker dye (Invitrogen) according to the manufacturer’s protocol.
After 72 h of MMC cell culture in uncoated, Matrigel-precoated, and
PCgel-precoated 24-well TCPs, 106 p98 T cells (Green Cell
Tracker-labeled) were added. After 30 h of treatment, dead p98 T cells
were washed away from MMC cells using PBS. Dead MMC cells were stained
with SYTOX Blue nucleic acid stain (Invitrogen) according to the manufacturer’s
instructions. Cells were imaged using an inverted fluorescence microscope
(Nikon TE 300, Japan). To quantify the dead MMC cells after the treatment
with labeled p98 T cells, the signals from the images were quantified
through ImageJ. The dead cell percentage was calculated from the following
equation:
For SEM analysis, samples
were fixed with a 4% formaldehyde aqueous solution for 30 min at room
temperature. After the fixation and dehydration in a series of ethanol
washes (70, 85, 95, and 100%), the samples were dried with a critical
point dryer (Denton DCP-1, Cherry Hill, NJ). The samples were mounted
on SEM pin stub, sputter-coated with platinum, and then imaged with
a JSM-7000F SEM (JEOL, Tokyo, Japan).
our previous studies, a ratio of 100:1 for effector (p98 T cells)
to target (MMC cells) was selected for this study.25 (link),26 For fluorescence imaging, p98 T cells were labeled with Green Cell
Tracker dye (Invitrogen) according to the manufacturer’s protocol.
After 72 h of MMC cell culture in uncoated, Matrigel-precoated, and
PCgel-precoated 24-well TCPs, 106 p98 T cells (Green Cell
Tracker-labeled) were added. After 30 h of treatment, dead p98 T cells
were washed away from MMC cells using PBS. Dead MMC cells were stained
with SYTOX Blue nucleic acid stain (Invitrogen) according to the manufacturer’s
instructions. Cells were imaged using an inverted fluorescence microscope
(Nikon TE 300, Japan). To quantify the dead MMC cells after the treatment
with labeled p98 T cells, the signals from the images were quantified
through ImageJ. The dead cell percentage was calculated from the following
equation:
For SEM analysis, samples
were fixed with a 4% formaldehyde aqueous solution for 30 min at room
temperature. After the fixation and dehydration in a series of ethanol
washes (70, 85, 95, and 100%), the samples were dried with a critical
point dryer (Denton DCP-1, Cherry Hill, NJ). The samples were mounted
on SEM pin stub, sputter-coated with platinum, and then imaged with
a JSM-7000F SEM (JEOL, Tokyo, Japan).
3
Cell Tracker Green Labeling Kinetics
4
Monitoring Cell Death in U937 Cells
5
3D Collagen Gel Embedding of Fibroblasts
6
Lung Cancer Cell Migration Assay
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Lung cancer cell lines were plated at a density of 106 cells/mL, and supernatants were collected after 3 days. The supernatants were filtered with 0.2 µm membrane and stored at −20 °C. The isolated CD14+ cells were fluorescently labeled with CellTracker Green Dye (Life Technologies, Carlsbad, CA, USA) and 105 labeled cells were placed in the upper chamber of 5 µm pore transwell inserts (Corning). Warmed supernatants from lung cancer cell lines were placed in the bottom chamber. Real-time phase and fluorescence live cell imaging was used to follow CD14+ cell migration, and object counts were obtained every 2 h (Essen Bioscience, IncuCyte FLR).
7
Cell Tracking for Cell-in-Cell Structures
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Cell lines were stained with CellTracker™ Green dye (Invitrogen) or CellTracker™ Red dye (Invitrogen) for 30 minutes at 37℃ in the absence of serum and washed with PBS three times. The cell-in-cell structures were counted using fluorescence microscopy.
8
Cell-in-cell Structure Isolation
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EBV-negative CNE-2 cells pre-stained with 1 μM CellTracker Red dye (Invitrogen) were co-cultured with GFP-Akata cells pre-stained with 2.5 μM CellTracker Green dye (Invitrogen) at 1:10 ratio for 12 h. After washing with pre-cold PBS for three times to remove free GFP-Akata cells, cells were trypsinized to a single-cell suspension and resuspended with PBS containing 10% FBS. Cells were sorted immediately on FACS Aria II (Becton Dickinson, USA), and double-labeled cells with heterotypic cell-in-cell structures were collected by centrifugation for 5 min at 500× g and plated on 96-well plates. Cells were selected in culture medium containing 450 μg/ml G418 for 2 weeks.
9
Cell Migration on 3D Nanofiber Scaffolds
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To evaluate migration on 3D nanofibre scaffolds, 1 × 103 cells labelled with Cell Tracker Green dye (Invitrogen, Paisley, UK) were cultured as hanging drops as described (Del Duca et al, 2004 (link)). Spheroid aggregates were transferred into wells of aligned poly-ɛ-caprolactone nanofibre-coated culture plates (Nanofibre solutions, Columbus, OH, USA), which allows cell migration on a 3D scaffold stimulated by topographical cues (Johnson et al, 2009 (link)). Spheroids were incubated at 37 °C in growth medium±20 mM LiCl or 5 μM BIO in triplicate and migration was monitored for 72 h using the EVOS imaging system and quantified using Image J software.
10
Capillary Sprouting Assay with iPS-Derived Macrophages
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The capillary sprouting assay was performed as previously described previously (Schulz et al., 2012 (link)). HDLECs were incubated in a 15 ml conical tube in presence of cytodex 3 beads (Sigma-Aldrich) at a ratio of 400 cells per bead in EGM-2MV medium (Lonza) for 4 h at 37°C with shaking every 20 min. Beads were then incubated in a cell flask at 37°C for 48 h in EGM-2MV medium. The HDLEC-coated beads were subsequently collected and labelled with 2 μM Cell Tracker Green dye (Invitrogen) for 30 min, embedded in 1 mg/ml Type I collagen hydrogel (Advanced Bio-Matrix) containing 2 μM D-erythro-sphingosine-1-phosphate (Avanti Polar Lipids), and cultured in black clear-bottom 96-well plates (Perkin-Elmer) in the presence or absence of 10,000 human iPS-derived macrophages-RFP per well. After 60 min of incubation, a 1:1 mix containing EGM2-Incomplete (EGM2 media without supplementation with EGF, FGF2 and VEGF-A; Lonza) and macrophage media (v/v) was added and the beads incubated for 2 days at 37°C. The beads were then fixed with 4% PFA for 30 min and washed twice with PBS. Each well was imaged with a Zeiss LSM 780 scanning confocal microscope and the number of sprouts per bead calculated.
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