Cell distribution in alginate hydrogel was identified using CellTracker™ Fluorescent Probes (Thermo Fisher Scientific) according to the manufacturer’s protocol. Briefly, the dye vial was thawed at room temperature before opening and the dye product was dissolved in DMSO to a final concentration of 10 mM. Serum-free medium was added to the dissolved dye vial and the solution was warmed to 37 °C. The prepared cells were harvested by centrifugation and aspiration of the supernatant. The cells were re-suspended in a pre-warmed solution (epithelial cells in the red-dye vial and bMSC in the green-dye vial) and incubated for 30 min at 37 °C in a 5% CO2 incubator. Thereafter, centrifugation and removal of the supernatant were performed again and the cells obtained were used in the 3D bioprinting as previously described. The manufactured artificial tracheas were observed under a light microscope with appropriate optical filters according to the color of the dye.
Celltracker fluorescent probes
Manufactured by Thermo Fisher Scientific
Sourced in United States
CellTracker™ Fluorescent Probes are a set of cell-permeant, fluorescent dyes that are used to monitor and track cells. These probes can be used to label live cells and can be detected using fluorescence microscopy or flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer a1, by Zeiss (3 mentions)
Anti cxcr2, by R&D Systems (2 mentions)
Qtracker cell labeling kit, by Thermo Fisher Scientific (2 mentions)
Verteporfin, by Merck Group (2 mentions)
Vybrant cfda se cell tracer, by Thermo Fisher Scientific (1 mentions)
Nis elements software, by Nikon (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Leica dmi8 microscope, by Leica camera (1 mentions)
Orca flash4.0 v3 digital cmos camera, by Hamamatsu Photonics (1 mentions)
Green cmfda, by Thermo Fisher Scientific (1 mentions)
Laminin ln521, by BioLamina (1 mentions)
Celltracker orange cmra, by Thermo Fisher Scientific (1 mentions)
Dcfh diacetate dcfhda kit, by Beyotime (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Te2000 u florescence microscope, by Nikon (1 mentions)
Eclipse ti s, by Nikon (1 mentions)
Celltracker red cmtpx dye, by Thermo Fisher Scientific (1 mentions)
Celltracker green cmfda dye, by Thermo Fisher Scientific (1 mentions)
Matrigel solution, by Corning (1 mentions)
Inverted microscope, by Olympus (1 mentions)
16 protocols using celltracker fluorescent probes
1
Cell Labeling for 3D Bioprinting
2
Visualizing Schistosome-Macrophage Trogocytosis
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The transfer of membrane from schistosomula to macrophages was detected by laser scanning confocal microscope. Schistosomula were labeled with Vybrant® CFDA SE Cell Tracer (2 µM, ThermoFisher, USA) for 15 min at 37 °C, and after discarding the staining solution, incubated in complete medium for 30 min. After washing with PBS, the parasites were incubated together with macrophages of mice or M. fortis labeled with Cell Tracker™ fluorescent probes (8 µM, ThermoFisher, USA) at an E:T ratio of 2000:1 in the presence of 5% mouse or M. fortis serum. In the M. fortis macrophage culture system, PP2 (20 µM), cytochalasin D (10 µM) or FK506 (10 ng/mL) were additionally added according to the purpose of the experiments. After the incubation for 12 h, samples were fixed with stop buffer containing 0.4% PFA and observed on a Nikon A1R N-SIM N-STORM microscope (Nikon, Japan). The images were acquired by Z-stack spanning. Data were then deconvolved using NIS-Elements software (Nikon, Japan), processed and a 3D reconstruction carried out. The percentage of cells positive for trogocytosis and the mean fluorescent intensity/single cell were analyzed using Imaris 8.4 software (Bitplane).
3
Quantifying Oxidative Stress in Oocytes
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The amount of ROS generated in the oocytes was evaluated using a dichlorofluorescin diacetate (DCFH-DA) assay kit (S0033S; Beyotime Biotechnology Inc., China). Briefly, oocytes were incubated in a medium containing an oxidation-sensitive fluorescent probe (DCFH-DA) at 37 °C for 30 min in the dark and then washed three times with phosphate-buffered saline (PBS). Upon transfer to a glass medium, the oocytes were observed under a laser scanning microscope (LSM 800; Zeiss, Germany). Fluorescent photographs of the oocytes were obtained using single-layer scanning, and the image acquisition parameters were consistent for all examined oocytes. The fluorescence intensity of each oocyte was measured using the ZEN software (Carl Zeiss IMT Co., Ltd., Germany).
Glutathione (GSH) levels in oocytes were assessed using CellTracker™ Fluorescent Probes (C12881; Thermo Fisher Scientific, USA). Each group of tested oocytes was stained with 10 μM CellTracker for 30 min at 37 °C in the dark and subsequently washed with PBS to remove surface fluorescence. The fluorescence intensity was measured using a laser scanning confocal microscope. The acquired photographs were analyzed using the ZEN software to measure the staining brightness of each oocyte.
Glutathione (GSH) levels in oocytes were assessed using CellTracker™ Fluorescent Probes (C12881; Thermo Fisher Scientific, USA). Each group of tested oocytes was stained with 10 μM CellTracker for 30 min at 37 °C in the dark and subsequently washed with PBS to remove surface fluorescence. The fluorescence intensity was measured using a laser scanning confocal microscope. The acquired photographs were analyzed using the ZEN software to measure the staining brightness of each oocyte.
4
PBMC Adhesion on PDMS Textures
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PDMS surface replicas mimicking various textures, placed in the bottom of a polystyrene 24-well chamber, were first sterilized under UV light. PBMCs, pre-stained with CellTracker Fluorescent Probes (Thermo Fisher Scientific Inc.), were seeded onto these surfaces. In experiments involving bacterial interaction, a suspension of S. epidermidis (strain ATCC 12228, OD = 0.1) in Tryptone Broth was added to the wells and incubated for 24 h at 37°C, after which the bacterial medium was replaced with the PBMC solution. Imaging was performed on a DMI8 Leica microscope, utilizing a 20× air objective and maintained under climate control at 37°C. For each experimental condition, 10 images across different vertical planes were captured using an ORCA-Flash 4.0 V3 Digital CMOS camera (Hamamatsu). Time-lapse imaging was conducted at 10-min intervals for up to 5 h, employing Metamorph (v7.10.1.161) for image acquisition.
5
Migration of CD271+ Cells Toward Islets
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Since boundary cap NCSCs are known to migrate toward islets, the migration behavior of the CD271+ cells was evaluated. CD271+ cells were co-cultured with either human islets or human ICC derived from pluripotent stem cells. A drop of approximately 50,000 cells and 2–3 islets were placed on opposite sides of a coverslip placed in a 4-well plate at a distance of approximately 1000 µm. The cells were cultured in medium suitable for islet culture. Coverslips were pretreated with Laminin LN521 (BioLamina AB, Sundbyberg, Sweden) for 2 h at 37 °C in 5% CO2 and 95% air. To visualize the cells, they were stained with CellTracker Fluorescent Probes (1:1000; Green CMFDA; Thermo Fisher Scientific). Images were taken on days 1–7 of co-culture, and the distance between cells and islets or ICC was measured. Experiments were repeated with three biological replicates, and the migration capacity toward human islets and ICC was compared and evaluated by measuring the distance to the CD271+ cells.
6
Cell Morphology Analysis Protocol
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For the cell morphological analysis, cell counts were determined by staining the cytoplasm using CellTracker fluorescent probes (Thermo Fisher Scientific). Cell morphology was then evaluated using ImageJ (National Institutes of Health, Bethesda, MD, USA), including the area (A), circularity (4πA/perimeter2), and aspect ratio (long-axis length/short-axis length, under Fit Ellipse mode). Besides, number of actin stress fibers was analyzed based on the intensity calculated by ImageJ software.
7
Fluorescent cell labeling protocol
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Cells that had reached 70–90% confluence were stained using the following CellTracker™ fluorescent probes (ThermoFisher Scientific, UK): CellTracker™ Green CMFDA, CellTracker™ Orange CMRA, and Cell Tracker™ Deep Red. Cells were stained following the manufacturer’s instructions. Briefly, anhydrous dimethyl sulfoxide (DMSO) was added to the lyophilised product to create 10 mM stock solutions of Green CMFDA and Orange CMRA dyes, and 1 mM stock solutions of the Deep Red tracker dye. Next, 20 µM working solutions of the Green and Orange dyes were obtained by adding the appropriate volume of stock solution to the specific growth medium. Due to the high fluorescent signal obtained from the Deep Red dye, the working concentration used was 1 µM.
Cells in culture flasks had media removed and were incubated at 37 °C/5% CO2/95% humidity with the dyes for 30–45 minutes. The CellTracker™ working solutions were then removed, and cells were washed with 5 mL 1× PBS twice, before continuing appropriate experimental procedures.
Cells in culture flasks had media removed and were incubated at 37 °C/5% CO2/95% humidity with the dyes for 30–45 minutes. The CellTracker™ working solutions were then removed, and cells were washed with 5 mL 1× PBS twice, before continuing appropriate experimental procedures.
8
Intracellular ROS and GSH Measurement
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A DCFH diacetate (DCFH-DA) kit (Beyotime Biotechnology, S0033, China) and CellTracker™ fluorescent probes (Thermo Fisher Scientific, C12881, China) were used to examine the level of intracellular ROS and GSH levels during oocyte maturation. The oocytes were incubated in PBS-PVA containing 10 μM 2′,7′-DCFH or 10 μM 4-CMF2HC for 15 min. After washing the oocytes three times in PBS-PVA, images were captured using a fluorescence microscope (Nikon, Tokyo, Japan).
9
Co-encapsulation of Leukemia and Cancer Cells
10
MDA-MB231 Cell Population Isolation
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