Fe-PEI NPs were allowed to aggregate for 24 h in DMEM in the presence of the fluorescent molecule DiI (1′-Dioctadecyl-3,3,3′,3′-Tetramethylindocarbocyanine Perchlorate Molecular Probes D-282, 30 µg/mL) either on ultra-low adherence (ULA) plates (that do not allow the adhesion of aggregates) or onto cell culture-treated chamber slides (in adherent conditions). Then, the aggregates were washed to remove not-entrapped fluorescent molecules and further used for the interaction with cells. To this aim, MSC suspension was seeded (at a density of 10,000 cells/cm2) into cell culture chamber slides (Lab-Tek II CC2 chamber slide system, Nunc, Rochester, NY, USA) either directly onto adhered Fe-PEI aggregates or in the presence of the suspension of Fe-PEI aggregates pre-formed in ULA conditions. The incorporation of DiI into MSCs was evaluated after 24 h. Briefly, cells were fixed with 4% PFA for 10 min at room temperature, followed by staining with Oregon Green-labelled WGA conjugate (wheat germ agglutinin, Molecular Probes, Eugene, OR, USA, 1/200 dilution) and Hoechst 33,258, for 10 min at room temperature. Washed slides were mounted with Prolong antifade reagent and examined by confocal microscopy (TCS-SP5 from Leica).
D 282
Manufactured by Thermo Fisher Scientific
Sourced in United States
The D-282 is a laboratory instrument designed for the measurement of dissolved oxygen (DO) concentration in aqueous solutions. It provides accurate and reliable DO measurements, enabling users to monitor and analyze the oxygen content in various liquid samples.
Automatically generated - may contain errors
Lab products found in correlation
M2bio, by Zeiss (2 mentions)
Lab tek 2 cc2 chamber slide system, by Thermo Fisher Scientific (1 mentions)
Tcs sp5, by Leica camera (1 mentions)
Confocal microscope, by Leica camera (1 mentions)
H3570, by Thermo Fisher Scientific (1 mentions)
Plan apo 60x oil objective, by Nikon (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
Dako fluorescent mounting medium, by Agilent Technologies (1 mentions)
Nis elements software, by Nikon (1 mentions)
Effectene transfection reagent, by Qiagen (1 mentions)
Dma 1511, by Narishige (1 mentions)
Vt1000s vibratome, by Leica Microsystems (1 mentions)
M2 microscope, by Zeiss (1 mentions)
11 protocols using d 282
1
Nanoparticle Uptake by Mesenchymal Stem Cells
2
Visualizing Murine Vascular Network
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Blood vessels were directly labeled by using an aqueous solution containing 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) (D-282, Invitrogen/Molecular Probes; 42,364, Sigma-Aldrich) [32 (link)]. Sedated mice were sacrificed by CO2 asphyxiation, followed by cervical dislocation. The abdominal cavity was opened via a transverse incision and the distal abdominal aorta was exposed. The proximal aorta was clamped in order to block the flow to the upper body. The Dil solution was injected into the distal aorta using the perfusion device (consisting of two three-way stopcocks, a 30-gauge butterfly needle and three 10-ml syringes). The perfusion order included: 1) 5 ml of PBS at the rate of 1–2 ml/min; 2) 5–10 ml of the DiI solution at the rate of 1–2 ml/min; and 3) 5–10 ml of the fixative at the rate of 1–2 ml/min. After perfusion, colon tissue was harvested and kept in 4% paraformaldehyde for 48 h. Stained and fixed, vessels were visualized with a Leica confocal microscope.
3
Tracing neuronal pathways with DiI
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Embryonic mouse brains or human brain slices were fixed with 1.5% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). DiI crystal (1,1′-dioctadecyl-3,3,3′,3′-tetramethylin-docarbocyanine perchlorate; D-282, Molecular Probes) was applied to the hypothalamic pia surface of the fixed mouse brains (E13.5 or E15.5) and human slices (GW11), which was allowed to diffuse throughout the tissue in PBS for 2–7 days at room temperature (RT). Labeled brains or slices were vibratome-sectioned for imaging analysis.
4
Localization and Quantification of Prestin
5
Dye-Filling Assay for Nematode Neurons
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A dye-filling assay was performed using an established protocol58 (link). Briefly, worms were washed off the culturing plate with M9 buffer (3 g l−1 KH2PO4, 6 g l−1, Na2HPO4, 5 g l−1 NaCl and 1 mM MgSO4), collected by centrifugation at 500 g for 1 min, washed once with M9 buffer, and then incubated in diluted DiI dye (Molecular Probes, D-282; 1:200 dilution in M9 buffer of the 2 mg ml−1 stock solution in dimethyl formamide) for 1 h at room temperature. After incubation, worms were washed at least three times with M9, transferred to an NGM plate without a bacterial lawn, and observed under a fluorescence microscope (Zeiss, M2Bio).
6
Multimodal Cortical Recording Procedure
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After animals were anesthetized with 1.5 g/kg urethane, the animal was placed in a stereotaxic frame (Narishige) and body temperature was retained at 37°C using a feedback temperature controller (40-90-8C, FHC). After incision, the bone above the left sensorimotor cortices (0–1 mm posterior from the bregma, 0–1 mm lateral from the midline) was removed and the cavity was filled with warm saline during the entire recording session. The μLED probe was slowly inserted into the cortex with 10° angle to the normal and penetrated 1.5 mm. A 32 channel silicon-based optrode (A1 × 32–10 mm-50-177-A32OA, NeuroNexus Technologies) was inserted slowly (~2 μm/sec) and penetrated up to 1.25 mm with a motorized manipulator (DMA-1511, Narishige). The distance between the μLED probe and optrode was between 200–500 μm at the cortical surface. Several penetrations were made for each animal. For histological verification of tracks, the rear of the probe was painted with DiI (D-282, ~10% in ethanol, Molecular Probe; Sakata and Harris, 2009 (link)).
7
Calcein-AM Dye Transfer Assay
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The calcein-AM dye transfer assay was performed according to a previously described method (Czyz et al, 2000 (link)). Briefly, donor melanoma cells were loaded with 1 μM calcein-AM (C-1359; Sigma-Aldrich) and acceptor melanoma cells were loaded with Dil (15 μg ml−1; D-282; Molecular Probes, Eugene, OR, USA) for 30 min at room temperature. After thoroughly washing with PBS, both cell types were cocultured at a 1 : 1 ratio (calcein+-donor cells : Dil+-acceptor cells) for 60 min at 37 °C. Calcein transfer from the donor to the acceptor cells was evaluated by flow cytometry, determining the percentage of calcein+ cells among the Dil+ cells. Calcein transfer assays were carried out in the presence of the Cx43-specific mimetic peptide 1848, control peptide gap20 or P5.
8
In Vivo Mouse Vascular Labeling
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Mouse blood vessels were directly labeled in vivo in anesthetized mice by live perfusion using a specially formulated aqueous solution (7 ml/mouse) containing DiI (D-282; Invitrogen/Molecular Probes), which incorporates into EC membranes upon contact, and was administered via direct intra-cardiac injection before animal euthanasia as previously reported (Li et al, 2008 (link); Shao et al, 2011 (link)). 7 ml of fixative (4% paraformaldehyde) was injected after Dil perfusion, and the entire wound tissue was harvested. The vascular network was visualized by scanning the entire wound tissue to a thickness or depth of 200 μm, using laser scanning confocal microscopy (Vibratome [VT1000S; Leica Microsystems]). Vessel density was quantified assessing total number of red Dil-labeled vessels normalized to the entire scanned wound area, using ImageJ software.
9
DiI Labeling of Mrgprd+ Neurons
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DiI (Molecular Probes, Cat# D282, 1 µL, 0.2 mg/mL dissolved in DMSO then diluted 1:5 with PBS) was subcutaneously injected in the plantar hind paw or shaved ventral proximal hind limb of 4pw MrgprdEGFPf mice (each mouse received DiI at both locations on opposite sides, and side-location combinations were alternated between mice). 7 days after injection, mice were perfused and skin and L3-L5 DRGs were dissected. Skin was post-fixed and mounted in PBS for imaging. DRGs were post-fixed, cryoprotected and then serially cryosectioned through the whole ganglion, and sections were mounted and imaged for quantification.
10
Amphid Neuron Staining with DiI
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Amphid neurons were stained with DiI (D-282, Molecular Probes) as described (45 (link)) with slight modifications. Briefly, well-fed young adult animals were collected from 6-cm plates, washed twice with M9 buffer, and incubated in 1 ml of M9 buffer containing 5 μl of DiI [dilution of 1:200 of stock (2 mg/ml)] overnight at room temperature. After incubation, the animals were washed three times with M9 buffer and transferred to NGM plates seeded with E. coli OP50. After incubating for 1 hour, the animals were mounted on agarose pads with 50 mM sodium azide and imaged using a Zeiss M2 microscope with a mechanized stage, Zen software, and Texas red filters.
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