Bone marrow cells were obtained from the tibia and femur of mice. Lineage-positive (Lin+) cells were depleted by magnetic-activated cell sorting using an APC-conjugated mouse lineage antibody cocktail (BD Pharmingen, San Diego, CA, USA) and anti-APC microbeads (Miltenyi Biotec, Auburn, CA, USA). After magnetic-activated cell sorting purification, the purity of Lin− cells was >95% in all experiments. For in vivo and in vitro donor cell tracking experiments, Lin− cells were labeled with PKH26 (Sigma-Aldrich, St Louis, MO, USA) or Vybrant DiI (Molecular Probes, Eugene, OR, USA) and stained with anti-Sca1 and anti-c-Kit antibodies (BD Pharmingen) and sorted using BD FACSAriaIII (BD Bioscience, San Jose, CA, USA). The purity of each sorted population was >99%.
Vybrant dii
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Vybrant DiI is a fluorescent dye used for labeling cell membranes. It is a long-chain carbocyanine dye that can be incorporated into the lipid bilayer of the cell membrane, allowing for the visualization and tracking of labeled cells.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1, by Zeiss (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Vybrant dio, by Thermo Fisher Scientific (3 mentions)
Ufc901008, by Thermo Fisher Scientific (2 mentions)
Spectramax id5, by Molecular Devices (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Syto rnaselect, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Huvecs, by Lonza (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Vybrant did, by Thermo Fisher Scientific (2 mentions)
Matrigel, by Corning (2 mentions)
Flow count fluorosphere, by Beckman Coulter (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
N2 supplement, by Thermo Fisher Scientific (2 mentions)
Glutathione sepharose 4b beads, by GE Healthcare (2 mentions)
Bicinchoninic acid bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
40 protocols using vybrant dii
1
Isolation and Purification of Mouse Hematopoietic Stem Cells
2
Visualizing DRG-NK Cell Interactions
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Adult or embryonic mouse DRG (103 and 8x103 neurons, respectively) cultured for one day on PDL and laminin-coated glass-bottom Petri dishes were fluorescently labeled with Vybrant DiI (Molecular Probes, cat no. V-22886) or loaded with the Ca2+ indicator rhodamine 3-AM (Molecular Probes, cat no. R10145) according to the manufacturer’s instructions. NK cells previously isolated from NKp46-YFP mice and stimulated IL-2 (1000U/ml) for 48 h were suspended in neurobasal media and seeded onto the coverslip (2.5x105 cells per dish). Dishes containing DRG-NK co-cultures were immediately transferred to a confocal microscope (LSM 700, Zeiss) and maintained in a humidified atmosphere at 37°C and 5% CO2 (Live Cell Instruments, Seoul Korea). A time series of single z section images (512 × 512) were acquired using a multitrack setting (488 nm and 555 nm fluorescence emission and differential interference contrast (DIC) bright-field) at 30-60 s intervals and multiple positions under the control of Definite Focus. Images were acquired up to 3h and exported as a sequential time-lapse in AVI format.
3
Live Imaging of Cell Spreading on Micropatterns
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Cells were seeded in petri dishes with microcontact printed substrates. After 20 min of incubation, cells were rinsed with phosphate buffered saline (PBS) to wash out non-adhered cells and fresh DMEM was added.
We used a Zeiss Axio Observer.Z1 equipped with a Heating Unit XL S, a Temp Module S, a Pecon Heating Insert P S1 and a CO2 Module S to generate physiological conditions of 37 °C and 5 % CO2 for live imaging. Phase contrast microscopy, using a 10x objective with a numerical aperture of 0.25, enabled a stable focus for long-term experiments and was used for all experiments of this study, but the ones carried out using TIRF illumination.
For TIRF microscopy, cells were stained with Vybrant DiI (Molecular Probes). A laser of 561 nm wavelength (part of a Zeis TIRF system) and a 100x objective with a numerical aperture of 1.46 were used. TIRF was used for monitoring the spreading phase of the cells and especially for a higher resolution of dynamics of actin bundles in the late spreading phase.
We used a Zeiss Axio Observer.Z1 equipped with a Heating Unit XL S, a Temp Module S, a Pecon Heating Insert P S1 and a CO2 Module S to generate physiological conditions of 37 °C and 5 % CO2 for live imaging. Phase contrast microscopy, using a 10x objective with a numerical aperture of 0.25, enabled a stable focus for long-term experiments and was used for all experiments of this study, but the ones carried out using TIRF illumination.
For TIRF microscopy, cells were stained with Vybrant DiI (Molecular Probes). A laser of 561 nm wavelength (part of a Zeis TIRF system) and a 100x objective with a numerical aperture of 1.46 were used. TIRF was used for monitoring the spreading phase of the cells and especially for a higher resolution of dynamics of actin bundles in the late spreading phase.
4
EV Labeling and Liposome-EV Fusion Assay
5
Hippocampal Neuron EV Uptake Assay
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Cultured hippocampal cells were incubated with hMSC-EVs (6 × 108 particles) for 22 h after addition of vehicle (PBS containing 2% DMSO) or AβOs (500 nM) for 2 h. In uptake experiments, we employed hMSC-EVs derived from hMSCs that were double-labeled with SYTO RNASelect (for RNA labeling) and Vybrant DiI (for membrane labeling) (both from Molecular Probes). Cells were fixed with 4% paraformaldehyde/4% sucrose solution for 15 min at 4 °C. Cell nuclei were labeled with DAPI. Images were acquired on a Zeiss LSM 510 confocal microscope.
For immunocytochemistry, fixed cells were blocked with 4% bovine serum albumin at room temperature. Primary antibodies used were rabbit anti-MAP 2 (1:200; Santa Cruz), mouse anti-GLUA1 (1:200; MAB2263, Santa Cruz), and guinea pig anti-GLT-1 (1:400; AB1783, Millipore) and were incubated overnight at 4 °C. For labeling with mouse anti-synaptophysin (1:1000; Vector Labs) and rabbit anti-PSD-95 (1:200; Santa Cruz) antibodies, cells were permeabilized with 0.1% Triton X-100 (Merck) for 5 min at room temperature before blocking with 10% horse serum for 1 h. After incubation with primary antibodies, cells were washed with PBS and incubated with Alexa Fluor-conjugated secondary antibodies (1:1000; Thermo Fisher) for 2 h at room temperature. Images were acquired on a Zeiss Axio Observer Z1 microscope or a Nikon C2 confocal microscope.
For immunocytochemistry, fixed cells were blocked with 4% bovine serum albumin at room temperature. Primary antibodies used were rabbit anti-MAP 2 (1:200; Santa Cruz), mouse anti-GLUA1 (1:200; MAB2263, Santa Cruz), and guinea pig anti-GLT-1 (1:400; AB1783, Millipore) and were incubated overnight at 4 °C. For labeling with mouse anti-synaptophysin (1:1000; Vector Labs) and rabbit anti-PSD-95 (1:200; Santa Cruz) antibodies, cells were permeabilized with 0.1% Triton X-100 (Merck) for 5 min at room temperature before blocking with 10% horse serum for 1 h. After incubation with primary antibodies, cells were washed with PBS and incubated with Alexa Fluor-conjugated secondary antibodies (1:1000; Thermo Fisher) for 2 h at room temperature. Images were acquired on a Zeiss Axio Observer Z1 microscope or a Nikon C2 confocal microscope.
6
Single-Cell Live Imaging of Cardiac Cells
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For single cell live imaging, MFBs and CMCs were stained with Vybrant™ DiI (Molecular Probes). Cells were washed twice with HBSS before being stained with Vybrant™ DiI (5 μl of stock solution diluted in 1 ml HBSS) for 20 min in the incubator. Thereafter, cells were washed three times with HBSS to prevent non-specific binding of the dye and reduce background fluorescence.
Imaging of CMC strands with endogenous or coating MFBs was performed on immuno-stained preparations. Pattern-growth cultures were washed twice with HBSS and fixed with 2% paraformaldehyde for 10 min. After a second washing step, strands were incubated for 20 min with a blocking buffer (PBS containing 20% goat serum) followed by a 2 h exposition to anti α-SMA antibodies (mouse monoclonal, Thermo Fisher) dissolved in PBS containing 1% goat serum and 0.15% Triton X-100. After washing, preparations were incubated for 20 min with a goat anti-mouse AlexaFluor 488 secondary antibody (Molecular Probes). Preparations were further washed and finally mounted. All steps were carried out at room temperature.
Imaging of CMC strands with endogenous or coating MFBs was performed on immuno-stained preparations. Pattern-growth cultures were washed twice with HBSS and fixed with 2% paraformaldehyde for 10 min. After a second washing step, strands were incubated for 20 min with a blocking buffer (PBS containing 20% goat serum) followed by a 2 h exposition to anti α-SMA antibodies (mouse monoclonal, Thermo Fisher) dissolved in PBS containing 1% goat serum and 0.15% Triton X-100. After washing, preparations were incubated for 20 min with a goat anti-mouse AlexaFluor 488 secondary antibody (Molecular Probes). Preparations were further washed and finally mounted. All steps were carried out at room temperature.
7
EV Labeling and Liposome-EV Fusion Assay
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EVs aliquots containing 2×108 EVs purified from human plasma samples were resuspended in 1 mL PBS containing 5 μL Vybrant DiI (Molecular Probes, V-22885) and 5 μL DiD (Molecular Probes, V-22887) and incubated at room temperature for 20 min, then filtered three times with a 100 kDa centrifugal filter unit (UFC901008, Thermo Fisher Scientific) at 3000g for 20–30 minutes at room temperature to remove free dyes and concentrate EVs to a ~50 μL final volume. Liposome aliquots containing 2×108 or 2×109 liposomes in 50 μL PBS were mixed with EVs double-labeled with DiI and DiD, and liposome-EV fusion reactions were performed as described above. Fluorescent signal was excited with 480 nm laser and fluorescent emission spectrum was measured with SpectraMax iD5 (Molecular Device) microplate reader from 525 nm to 750 nm.
8
Apoptosis Induction in GFP-RAW and MC Cells
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Apoptosis was induced in vitro by incubation of either GFP-RAW cells or vybrant DiI (Molecular Probes Life Technologies, Eugene, OR, USA) stained MC with 50µM cisplatin (cis-diamineplatinum (II) dichloride from Sigma-Aldrich, Munich, Germany) for 24 hours at 37°C similar as described before [24] . For vybrant DiI dye staining, MC were seeded in culture dishes and were allowed to adhere overnight. Then, vybrant DiI dye was added (5µl/ml DMEM) for 10min at 37°C. Apoptosis was evaluated as described in [24] . Hoechst staining was used to analyze apoptosis via counting of chromatin-condensed cells. Trypan blue exclusion was used to determine viability (in average 80%).
9
Fluorescent Labeling of Cells
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Sample preparation followed a protocol we published previously42 (link). Briefly, nanosensor labeled cells were trypsinized and washed with 4 °C PBS once. Then cells were suspended at a density of 1 × 106/mL in membrane dye working solution (5 μM Vybrant DiI or DiB, Life Technologies) for 5 minutes at 37 °C. After washing 3× with cell culture medium, cells were re-seeded on fibronectin-coated cover slips for 20 minutes. Then cells were fixed with fresh-prepared chilled 4% paraformaldehyde in PBS for 10 minutes on ice. Later, NucBlue® Live cell stain was used to stain the nucleus before mounting onto microscope slides for visualization with confocal microscope LSM710 (Zeiss).
10
Long-term Cell Tracking with DiI
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For selected in vitro experiments, CPs were stained with the long-term cell trackers VyBrant diI (Life Technologies). DiI was diluted 1:1000 in PBS and incubated with confluent cells (adherent to the culture plate) for 5 minutes at 37°C and then on ice for an additional 15 minutes, in the dark. Cells were then washed with PBS, left to recover for 24 hours, and used for experiments.
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