SCLC and retinoblastoma cell lines transduced with YAP-GFP or control GFP viruses were pulsed with EdU (10 µm) for 20 min (Y79/ SHP77) or 30 min (WERI/H2171), harvested and fixed in 4% PFA for 10 min. After permeabilization, cells were stained for goat anti-GFP (1 hr), washed and stained using a donkey anti-goat Alexa488 secondary antibody (30 min). EdU was then labelled for 30 min using standard Click chemistry with an Alexa647-conjugated azide (Thermo Fisher). After washing cells were counterstained using the FxCycle violet DNA dye (Thermo Fisher) and analyzed on a Beckman Coulter Gallios flow cytometer and Kaluza analysis software.
Alexa647 conjugated azide
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa647-conjugated azide is a fluorescent dye used for labeling biomolecules. It consists of an azide functional group attached to the Alexa Fluor 647 dye. The azide group allows for covalent coupling to alkyne-containing compounds through click chemistry.
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Lab products found in correlation
2 protocols using alexa647 conjugated azide
1
Cell Proliferation Assay with YAP-GFP
2
Doxycycline-Induced PAR1 Nanoscale Visualization
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A549 cells were seeded in 35-mm N-STORM super-resolution microscope dishes and were grown to 60% confluency. Next, 0.2 μM doxy-yne was added to the cells and incubated for 4 h at 37°C in the presence of 5% CO2, followed by UV irradiation (365 nm) for 30 min. The cells were then fixed and permeabilized and 0.1 mM TBTA, 1 mM TCEP, 0.1 mM Alexa 647-conjugated azide (Thermo Fisher, USA) and 1 mM CuSO4 were added to the cells for 2 h for the click reaction. After the click reaction, the cells were incubated with rabbit polyclonal anti-PAR1 (CST, USA) in a humidified chamber overnight at 4°C and then were incubated with Cy3B-conjugated goat anti-rabbit secondary antibody for 2 h at room temperature, followed by seal slicing. The N-STORM super-resolution microscope (Nikon, Japan) was used to measure doxycycline and PAR1 co-localization on the nanoscale level.
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