Briefly, growth factor-depleted Matrigel was thawed overnight at 40 °C and mixed to homogeneity. Culture plates (48-well) were coated with 0.1 ml of Matrigel and allowed to gelatinize at 37 °C for 30 min. HUVECs or HUVECS and HTR8 cells (2.5 × 104 each), labeled with cell tracker Red CMTPX and Green CMFDA (Molecular Probes), respectively, were cultured on Matrigel in the presence of 10% normal pregnancy serum (NPS) containing A-1254 (10 μg/ml). The concentration of A-1254 was selected from a dose response studies described earlier11 (link). The endothelial cell tube formation was monitored and recorded after 12–14 hrs of incubation using the florescence microscopy at 4 x magnification (Nikon Eclipse TS 100 coupled with CCD camera) as described11 (link)33 (link). To evaluate the significance of the Notch/Dll pathway in A-1254-induced inhibition of angiogenesis, A-1254-induced disruption of three-dimensional tube formation by HUVECs was assessed in the presence or absence of different doses of γ-secretase inhibitor, L1790 (0, 1, 5, 10, 25 μM) (Sigma-Aldrich).
Green cmfda
Manufactured by Thermo Fisher Scientific
Sourced in United States
Green CMFDA is a fluorescent dye used for cell labeling and tracking in cell biology and biochemistry research applications. It is a cell-permeant dye that is non-fluorescent until it reacts with intracellular glutathione, resulting in a green fluorescent product. The core function of Green CMFDA is to provide a fluorescent marker for live-cell imaging and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Red cmtpx, by Thermo Fisher Scientific (9 mentions)
Celltracker deep red, by Thermo Fisher Scientific (2 mentions)
Celltracker red cmtpx, by Thermo Fisher Scientific (2 mentions)
Blue cmac, by Thermo Fisher Scientific (2 mentions)
Rosettesep human monocyte enrichment cocktail, by STEMCELL (2 mentions)
Balb c rag2 γc mice, by Thermo Fisher Scientific (2 mentions)
Nod scid il2rγ nsg mice, by Charles River Laboratories (2 mentions)
Ultra low attachment multiwell plates, by Corning (2 mentions)
6 well plastic plates, by Techno Plastic Products (2 mentions)
Eclipse ts100, by Nikon (1 mentions)
Lsm 510 meta confocal microscope, by Zeiss (1 mentions)
Celltrace violet, by Thermo Fisher Scientific (1 mentions)
Pkh26, by Merck Group (1 mentions)
Pkh67, by Merck Group (1 mentions)
Cytochalasin d, by Cayman Chemical (1 mentions)
Latrunculin a, by Cayman Chemical (1 mentions)
Celltracker red, by Thermo Fisher Scientific (1 mentions)
Confocal microscope, by Leica (1 mentions)
Vybrant cm dii, by Thermo Fisher Scientific (1 mentions)
Dulbecco s phosphate buffered saline dpbs 1x, by Biowest (1 mentions)
30 protocols using green cmfda
1
Inhibition of Angiogenesis by Notch Pathway Modulation
2
Confocal Microscopy of 3D Oral Mucosal Models
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One 3D oral mucosal model from each group was used for confocal microscopy. The models were first rinsed with PBS three times and then a mixture of fluorescent labels, red propidium iodide (PI), and green CMFDA (Molecular Probes) in serum-free medium, both at a final concentration of 5 µM, was added to the oral mucosal models from each group and incubated for 1 h, after which the medium was discarded and the models were rinsed with PBS and fixed with 10% formalin solution (Sigma-UK). The analysis was carried out using a Zeiss LSM510 Meta confocal microscope at the confocal imaging facility at the Kroto Institute, University of Sheffield, with a set of two lasers (green, 488 nm and red, 543 nm). The images were examined by two observers independently as described in Section 2.7.2 above.
3
In Vivo Platelet Tracking
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2 × 105 MKs, previously stained with Green CMFDA or CellTracker Deep Red (Molecular probes) and co-cultured or not with marrow cells, were injected i.v. in 200 μl PBS. Blood was harvested by tail vein sampling at indicated time points using heparinized capillary tubes. 1 μl was blood is diluted in 500 μl PBS in the presence of an anti-CD41 antibody. Presence of Green CMFDA or CellTracker Deep Red on circulating CD41+ platelets was evaluated by flow cytometry.
4
Cell Labeling and Biotinylation
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Latrunculin A and Cytochalasin D were purchased from Cayman Chemical. Ezrin inhibitor NSC668394 was from Calbiochem. Lipids cells strainers PKH67 and PKH26 were from Sigma. Protein strainers Green CMFDA, CellTracker Deep Red and Cell Trace Violet were from Molecular probes. Surface protein biotinylation kit was purchased from Pierce.
5
Three-Dimensional Cell Labeling Technique
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Cell TrackerTM fluorescent probes were used to demonstrate the three-dimensional positions of all three cell types. In this study, Cell TrackerTM Red CMPTX (C34552), Blue (C12881), and Green CMFDA (C7025) (Molecular Probes, Invitrogen) were used to label cells. After the cells were cultured in the appropriate medium, the medium was removed, and then pre-warmed (37 °C) Cell TrackerTM working solution (0.1 μM) that had been prepared in serum-free medium was gently added; the cells were incubated with this solution for 45 minutes at 37 °C. Then, the cells were trypsinized, centrifuged and seeded into the microfluidic device. After the cells in all three layers reached confluence, they were imaged under a fluorescence microscope.
6
Cell Tracking and Quantification
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CellTracker Red CMTPx (Molecular Probes) and Green CMFDA (Molecular Probes) were used to mark M1 and M2, respectively. Quantification was performed using Imaris (Bitplane) software. All the cells from a six-well plate were counted; each cell was identified as a color-coded dot. The total number of cells was determined counting the red (M1) or green (M2), then the number of red signals within the green cells was measured to establish engulfment events. The ratio M1/M2 was represented as the average of three independent experiments.
7
Cell Tracking for Coculture Imaging
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Substrates patterned
with fluorescently labeled proteins were imaged on a Nikon TE200 or
Nikon TE2000U microscope. For migration studies, cells were imaged
using brightfield microscopy. To visually identify distinct cell types
in patterned cocultures, cell types were labeled with the spectrally
distinct fluorescent dyes, CellTracker Red CMTPX and Green CMFDA (Molecular
Probes). For labeling, cells were incubated in 5 μM Cell Tracker
dyes for 30 min in serum-free media. Cells were then rinsed and incubated
in serum-containing media for at least 1 h.
with fluorescently labeled proteins were imaged on a Nikon TE200 or
Nikon TE2000U microscope. For migration studies, cells were imaged
using brightfield microscopy. To visually identify distinct cell types
in patterned cocultures, cell types were labeled with the spectrally
distinct fluorescent dyes, CellTracker Red CMTPX and Green CMFDA (Molecular
Probes). For labeling, cells were incubated in 5 μM Cell Tracker
dyes for 30 min in serum-free media. Cells were then rinsed and incubated
in serum-containing media for at least 1 h.
8
Coculture of Labeled Myoblasts and Myotubes
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Coculture of labeled myoblasts and myotubes has been described (Horsley et al., 2003 (link); Sohn et al., 2009 (link)). In brief, L6 myoblasts and nascent myotubes were labeled with 5 μM CellTracker red and green CMFDA (Molecular Probes), respectively, for 30 min at 37°C, washed with PBS twice, and incubated with fresh growth medium for another 30 min at 37°C. Labeled cells were trypsinized, plated in 12-well plates at equal cell numbers, and cocultured for another 24 h. Cells were then fixed with 3.7% formaldehyde, stained with DAPI, and visualized with a Leica confocal microscope. Fusion between myoblasts with nascent myotubes was analyzed in double-stained myotubes with ≥3 nuclei. More than 300 nuclei per sample were counted for analysis.
9
Cell Staining for 3D Co-culture Imaging
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Prior to cell harvesting, CRC cells, fibroblasts and endothelial cells were washed with PBS and incubated in serum free medium for 20 minutes supplemented with 40 µM blue CMAC, 5 µM Green CMFDA or 10 µM Red CMTPX (C2110, C7025 and C34552, Life Technologies), respectively. Afterwards, 3D co-cultures were established and fluorescent imaging was performed.
10
Labeling Tumor Cells for Tracking
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Tumor cells were grown to 70% confluence, washed with Dulbecco’s phosphate-buffered saline (DPBS) 1X (Biowest) and stained in a flask with lipophilic dyes—Vybrant CM-DiI (4 μl/ml in DPBS 1X), green CMFDA (1 μl/ml in DPBS 1X, 1 mM stock), or Deep Red Cell Tracker (1 μl/ml in DPBS 1X, 10 mM stock) (Life Technologies), for 10 min at 37 °C, in darkness. Cells were washed with DPBS and detached with 2 mM EDTA by scrapping. Cell suspension was collected to 1.5 ml eppendorfs, centrifuged at 250 × g, for 4 min at 4 °C, and resuspended in DMEM. Cell viability was assessed by trypan blue exclusion method, and cell number was determined by hemocytometer counting. Cells were resuspended in DPBS 1X to a final concentration of 0.25 × 106 cells/μl.
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