One day before the assay, NK cells were grown in culture in NK cell media supplemented with IL-2 (50 IU/mL) or IL-15 (10 ng/mL) for 12 to 18 hours, and neuroblastoma cells were plated at a density of 50,000 cells per well in 4-well glass bottom chamber slides (Ibidi). On the day of the assay, neuroblastoma cells were labeled with calcein-AM, and NK cells were labeled with CellTracker Orange (Thermo Fisher Scientific), according to manufacturer instructions. Hu14.18K322A (10 μg/mL) was added to the wells for 1 hour to allow binding to tumor cells. Dye-labeled NK cells were then added to the wells containing neuroblastoma cells. A C2 Nikon confocal microscope (Nikon) was used to perform live-cell imaging at a magnitude of 10X for 12 hours. Five different areas were imaged per condition. To quantify the imaging data, we extracted the green (8 bit) and red (16 bit) channels and segmented the green and red signal with FIJI software (12 (link)) and Weka Trainable Segmentation plug-in (13 ). We converted the segmented images into a binary mask and applied the 3D ROI manager plug-in to calculate contact area in μm and signal intensity (14 (link)).
Celltracker orange
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
CellTracker Orange is a fluorescent dye that can be used to label and track living cells. It is a cell-permeant dye that becomes fluorescent upon entering the cell and remains there, allowing for the visualization and monitoring of cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Celltracker green, by Thermo Fisher Scientific (23 mentions)
Celltrace violet, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Celltracker blue, by Thermo Fisher Scientific (3 mentions)
Hoechst, by Thermo Fisher Scientific (3 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (3 mentions)
Medimachine, by BD (3 mentions)
Celltracker red, by Thermo Fisher Scientific (3 mentions)
Calcein am, by Thermo Fisher Scientific (2 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (2 mentions)
Cell suspension reagent, by Standard BioTools (2 mentions)
Efluor 670, by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Cell tracker violet, by Thermo Fisher Scientific (2 mentions)
Mitotracker red, by Thermo Fisher Scientific (2 mentions)
70 μm nylon cell strainer, by BD (2 mentions)
Cellvue claret, by Merck Group (2 mentions)
Flow count beads, by Beckman Coulter (2 mentions)
92 protocols using celltracker orange
1
Live-cell Imaging of NK-Neuroblastoma Interaction
2
Phagocytosis of Apoptotic Thymocytes
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Thymi of C57BL/6 were harvested and passed through a 70 μm cell trainer. Thymocytes were labelled with cell tracker orange (ThermoFisher) according to manufacturer’s instructions, then rendered apoptotic via treatment with 1 μM dexamethasone (Sigma) for 17 hours (38 (link)). Murine BMDMs were pre-treated for 17 hours with iBET (0.5 μM) or DMSO control. Phagocytosis was assessed by flow cytometry, 4 hours after incubation of BMDMs with apoptotic thymocytes.
3
Adoptive Transfer of Fluorescent Naïve T Cells
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Experiments were performed with C57BL/6 mice (Jackson Laboratories), B6.Ubiquitin-GFP mice (Jackson Laboratories), B6.CCR7−/− mice (Jackson Laboratories) and B6.Cg-Tg(Itgax-Venus)1Mnz/J mice (Jackson Laboratories). Both female and male mice were used between 8 and 20 weeks of age. Breeding, maintenance, and use of animals used in this research conform to the principles outlined by the Institutional Animal Care and Use Committee (IACUC). The IACUC at the University of New Mexico approved the protocol for animal studies (protocol number 16-200497-HSC). Anesthesia via ketamine and xylazine was performed during mouse injections, and euthanasia was administered via isofluorane overdose followed by cervical dislocation. For blood vessel staining, DyLight 594 labeled Lycopersicon esculentum (tomato) lectin (Vector Laboratories) was used at a dose of 70 μg per mouse. To isolate naïve T cells, Pan T Cell Isolation Kit II (mouse, Miltenyi Biotec, 130-095-130) was used according to manufacturer’s instructions. To fluorescently label naïve T cells, CellTracker™Orange (5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine) (CMTMR) Dye (ThermoFisher Scientific, C2927) was incubated with naïve T cells at a final concentration of 5 μm at 37°C for 30 min before being washed. Labeled naïve T cells were then immediately adoptively transferred into recipient mice.
4
CTC Preparation and Staining
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CTC-rich blood samples were centrifuged at 300g for 10 min and concentrated to 70 μL. The cell staining was performed with the addition of the following markers and antibodies for 1 h: CellTracker Orange (CTO; Thermo Fisher Scientific C34551), Calcein AM (Thermo Fisher Scientific L3224), PTPRC antibody conjugated with Alexa Fluor 647 (BioLegend 304020), and CD31 conjugated with Alexa Fluor 647 (BioLegend 303111). To improve the viability and RNA quality of the cells, 15 μL of RPMI with 10% FBS (Gibco) and 3 μL of RNase inhibitor (Thermo Fisher Scientific N8080119) were also added. After incubation, 13 mL of PBS was added to dilute the staining reagents. The sample was spun down at 300g for 10 min and concentrated to 45 μL. To achieve optimal buoyancy in an integrated fluidic circuit (IFC), 45 μL of CTCs was mixed with 30 μL cell suspension reagent (Fluidigm 101-0434) to achieve 75 μL of cell mix.
5
Single-cell genomic analysis of circulating tumor cells
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The CellSearch-enriched CMCs were picked up by laser capture microdissection with the MMI CellCut system (Molecular Machines & Industries GmbH, Eching, Germany) mounted on an ECLIPSE Ti2 microscope (Nikon Corporation, Tokyo, Japan), subjected to Ampli1 WGA (Menarini Silicon Biosystems) according to the manufacturer’s instructions (with minor modifications), and finally sent to Menarini Silicon Biosystems for NGS analysis with the Ampli1 OncoSeek Panel, which is designed specifically to fit with the Ampli1 WGA protocol (Supplementary Material—Materials and Methods). To validate the entire workflow, 44 samples from different tumor cell lines in the form of spike-in samples or cells suspended in their medium were enriched by the CellSearch platform or simply stained in culture (CellTracker Orange, Thermo Fisher Scientific) to undergo microdissection at the single-cell or cluster level, and finally WGA. Additional samples were also subjected to Ampli1 OncoSeek analysis to validate the entire workflow.
When analyzing patient samples, a minimum of 5 cells per CellSearch cartridge were microdissected to increase the chance of a successful WGA reaction. White blood cells were collected together if the number of CMCs was <5. The quality of the WGA products was tested using the Ampli1 QC Kit (Menarini Silicon Biosystems) following the manufacturer’s instructions.
When analyzing patient samples, a minimum of 5 cells per CellSearch cartridge were microdissected to increase the chance of a successful WGA reaction. White blood cells were collected together if the number of CMCs was <5. The quality of the WGA products was tested using the Ampli1 QC Kit (Menarini Silicon Biosystems) following the manufacturer’s instructions.
6
Fluorescent Cell Labeling Techniques
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The fluorescent dyes 5(6 (link))-CFDA, SE (CFSE), (5-(and−6)-(((4-chloromethyl) benzoyl) amino) tetramethylrhodamine) (CMTMR, CellTracker orange) and Chloromethyl-coumarin (CMAC, CellTracker blue) were purchased from ThermoFisher (Basel, Switzerland) and the cell proliferation dye eFluor 670 was from eBioscience (San Diego, CA). mAb against PNAd (MECA-79) was from nanotools (Freiburg, Germany) and coupled to AlexaFluor-633 using a Protein Labeling Kit from Molecular Probes (Basel, Switzerland). Fc receptor blocking mAb (clone 2.4G2, 4.5 mg/mL) in FACS buffer (D-PBS supplemented with 1% milk powder and 0.1% NaN3) was produced in-house. T cells were fixed with 1% paraformaldehyde (PFA; Electron Microscopy Sciences, Lucerne, Switzerland) diluted in D-PBS. D-PBS supplemented with 10 mM EDTA was used for homogenizing LNs for flow cytometry experiments.
7
Adoptive Transfer of TCR Transgenic T Cells
8
Flow Cytometry and Microscopy for Cell-Cell Interactions
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For flow cytometry analysis, Lec2 CHO cells were labeled with CellTracker orange (CTO) following the manufacturer’s protocols (ThermoFisher Scientific), sialylated (as described above) and mixed with hSig15-expressing CHO cells that also express GFP in a ratio of 100:1 for 20−30 min on ice prior to analysis by flow cytometry. The GFP+ cell population was analyzed for CTO staining and interacting cells were defined as double stained. For fluorescence microscopy analysis, Lec2 CHO cells stained with CTO were sialylated as described above, mixed with hSig15-expressing CHO cells that also express GFP in a ratio of 100:1 for 20−30 min on ice prior to plating and incubation at 37 °C for 10 min to allow adherence. Plates were gently rinsed once with PBS and cell clustering was imaged by fluorescence microscopy. Images of each sample were analyzed for cell−cell interactions. The number of hSig15 CHO cells that were interacting with Lec2 CHO cells was counted and expressed as a percentage of the total number of hSig15 CHO cells analyzed.
9
Imaging of Cytotoxic NK Cell Conjugates
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YTS-CD16, NK92-CD16, or eNK cells co-cultured with S2, S2-IgG, S2-IC1, or S2-IC1-IgG cells for 15 min were adhered to poly-l -lysine–coated glass slides for 20 min at 37°C. The surrogate target S2 cells were previously labeled with CellTracker orange (Thermo Fisher Scientific). Fixation, permeabilization, and staining were performed as described (Banerjee et al., 2007 (link)). The reagents/antibodies were used in the following sequence for optimized results: (1) biotinylated monoclonal mouse anti-tubulin (Invitrogen); (2) streptavidin–Pacific blue (Invitrogen); and (3) FITC-conjugated mouse anti-perforin clone δG9 (BD). Slides were mounted with 0.15-mm coverslips (VWR) using ProLong AntiFade (Invitrogen). The image acquisition settings were as described in “Live-cell confocal microscopy,” and all transmitted light images were specifically from the focal plane of the MTOC of the NK cell, which may not have been ideal for those images, especially the conjugated target cell, and are thus provided for orientation only.
10
Lung Colonization Assay in Mice
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The lung colonization assay was examined using 6‐wk‐old female BALB/c nude mice (Clea). The cells were fluorescently labeled with CellTracker Green and CellTracker Orange (Thermo Fisher Scientific). Control cells and ZF21NT cells (1 × 105 each) were injected into the tail veins of nude mice, which were sacrificed 1 or 24 h later. Following lung dissection, fluorescently labeled cells were counted by confocal microscopy (Zeiss LSM 710). All mice were maintained under specific pathogen‐free conditions and the experiments were performed in accordance with the institutional animal care and use committee (Kanagawa Cancer Center).
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