The chicken embryo transplantation was performed as previously described [4 (link)]. Briefly, melanoma cells were treated with PAPPA-specific siRNAs or scrambled control siRNA as described and labelled with CM-DiO as per manufacturer's instructions (Invitrogen, USA). Cells were grown overnight in a hanging-drop fashion to allow the formation of aggregates. Fertile chicken eggs were incubated at 38°C for 48 hours prior to transplantation. Cell aggregates were harvested and carefully injected with a glass pipette into the trunk neural tube lumen of developing chicken embryos. The eggs were then sealed with adhesive tape and re-incubated for 2 days. After incubation, embryos were removed from the eggs and fixed with 4% paraformaldehyde and whole mounts or cross sections were analyzed for the localization of melanoma cells using a Lumar V12 Zeiss microscope.
Cm dio
Manufactured by Thermo Fisher Scientific
Sourced in United States
CM-DiO is a lipophilic carbocyanine dye used for fluorescent labeling of cell membranes. It passively diffuses into the lipid bilayer and can be used to stain live or fixed cells.
Automatically generated - may contain errors
4 protocols using cm dio
1
Chicken Embryo Transplantation of Melanoma Cells
2
Melanoma Cells Transplantation in Chicken Embryos
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Melanoma cells were treated with PXDN, NTN4 or GLIS3-specific siRNAs or scrambled control siRNA as described above and labelled with CM-DiO or Dil as per manufacturer's instructions (Invitrogen, USA). Cells were grown overnight in a hanging-drop fashion to allow the formation of aggregates. Fertile chicken eggs were incubated at 38°C for 48 hours prior to transplantation. Cell aggregates consisting of similar sized small aggregates from approximately 500–800 cells were harvested and carefully injected with a glass pipette into the trunk neural tube lumen of developing chicken embryos. The eggs were then sealed with adhesive tape and re-incubated for 2 days. After incubation, embryos were removed from the eggs and fixed with 4% paraformaldehyde and whole mounts were analyzed for the localization of melanoma cells using Lumar V12 Zeiss microscope as described previously [20 (link)]. Segments of embryos containing the melanoma cells were then washed in PBS, incubated overnight in 30% sucrose at 4°C, frozen and 20μm transverse sections cut using a cryostat. Sections were rinsed in PBS, mounted in fluorescent mounting media and imaged using a Zeiss Axio M1 microscope and AxioVision software.
3
Exosome Uptake by HUVECs Visualized
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Immunofluorescence staining was performed to confirm that HUVECs could take up exosomes. U87-MG cells were labeled with 3,3′-dihexadecyloxacarbocyanine perchlorate (CM-Dio; Invitrogen, Grand Island, NY, USA), according to the supplier's instructions. Exosomes derived from Dio-labeled U87-MG cells were routinely collected, and then HUVECs were incubated with 100 µg/ml of exosomes for 6 h. Next, HUVECs were rinsed with PBS and fixed with 4% paraformaldehyde at room temperature for 30 min and then pre-incubated with sodium borohydride (1 mg/ml in PBS) to decrease autofluorescence. The cells were then incubated overnight at 4°C with a CD31 primary antibody (mouse monoclonal anti-CD31, 1:100; Abcam) and then incubated for 1 h with a secondary antibody conjugated to Alexa Fluor 594 (1:200; Abcam). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, 0.5 µg/ml; Invitrogen). Images were obtained with a fluorescence microscope (Leica, Solms, Germany).
4
Melanoma Cell Xenotransplantation in Chick Embryos
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Melanoma cells were treated with THBS1-specific siRNAs or scrambled control siRNA as described and labelled with CM-DiO or Dil as per manufacturer's instructions (Invitrogen, USA). Cells were grown overnight in a hanging-drop fashion to allow the formation of aggregates. Fertile chicken eggs were incubated at 38°C for 48 hours prior to transplantation. Cell aggregates consisting of 50-400 cells were harvested and carefully injected with a glass pipette into the trunk neural tube lumen of developing chicken embryos. The eggs were then sealed with adhesive tape and re-incubated for 2 days. After incubation, embryos were removed from the eggs and fixed with 4% paraformaldehyde and whole mounts or cross sections were analyzed for the localization of melanoma cells using Lumar V12 Zeiss microscope.
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