This assay is based on a split-GFP system (pQCXIP-GFP1–10 and pQCXIP-GFP11) where GFP signals are produced upon cell fusion (Kodaka et al., 2015 (link)). The split GFP plasmids: pQCXIP-BSR-GFP1–10 and pQCXIP-GFP11 were a gift from Yutaka Hata (Addgene plasmid #68715, https://www.addgene.org/68715/ and #68716 https://www.addgene.org/68716/ ). Briefly, 200,000 Vero-TMPRSS2 cells were co-transfected with 500 ng pCAGGS- spike and 500 ng pQCXIP-GFP110 or pQCXIP-GFP11 using TransIT-LT1 (Takara; MIR2300). The next day, cells were detached, pooled and reseeded in black 96-well or 24-well plates (Greiner, M0562–32EA) at 25,000 and 200,000 total cells/well, respectively. After 24 h, cells were fixed with 4% paraformaldehyde in PBS for 10 min. To measure spike expression, cells were permeabilized with 0.1% Triton X-100 for 15 min and stained with primary antibody anti-S2 (Sino Biological, 40590-D001) and secondary Alexa-Fluor 594 goat anti-human antibody (Thermo Fisher, A-11014). DAPI was used to visualize the nucleus. Images of the whole wells were obtained and analyzed using the Celigo Image Cytometer (Nexcelom). GFP signals were normalized to spike expression and DAPI counts to represent fusion activity.
M0562 32ea
Manufactured by Greiner
The M0562–32EA is a laboratory equipment product. It is designed for use in scientific and research applications. The product's core function is to provide a specific capability or measurement for laboratory procedures, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
2 protocols using m0562 32ea
1
Quantifying SARS-CoV-2 Spike-Mediated Cell Fusion
2
Split-GFP Assay for Measuring Cell Fusion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
This assay is based on a split-GFP system (pQCXIP-GFP1-10 and pQCXIP-GFP11) where GFP signals are produced upon cell fusion (Kodaka et al., 2015 (link)). The split GFP plasmids: pQCXIP-BSR-GFP1-10 and pQCXIP-GFP11 were a gift from Yutaka Hata (Addgene plasmid #68715, https://www.addgene.org/68715/ and #68716 https://www.addgene.org/68716/ ). Briefly, 200,000 Vero-TMPRSS2 cells were co-transfected with 500 ng pCAGGS- spike and 500 ng pQCXIP-GFP1-10 or pQCXIP-GFP11 using TransIT-LT1 (Takara; MIR2300). The next day, cells were detached, pooled and reseeded in black 96-well or 24-well plates (Greiner, M0562-32EA) at 25,000 and 200,000 total cells/well, respectively. After 24 h, cells were fixed with 4% paraformaldehyde in PBS for 10 min. To measure spike expression, cells were permeabilized with 0.1% Triton X-100 for 15 min and stained with primary antibody anti-S2 (Sino Biological, 40590-D001) and secondary Alexa Fluor 594 goat anti-human antibody (Thermo Fisher, A-11014). DAPI was used to visualize the nucleus. Images of the whole wells were obtained and analyzed using the Celigo Image Cytometer (Nexcelom). GFP signals were normalized to spike expression and DAPI counts to represent fusion activity.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!