To detect CHX-mediated inhibition of protein synthesis, Gli36-mCherry cells in a 24-well plate were prepared in parallel to the EV-uptake and –RNA translation experiment as described above. At the indicated post-centrifugation time points, culture medium was replaced with methionine- and cysteine-free medium containing Click-iT® homopropargylglycine (HPG; Invitrogen) for 30 min, and the cells were washed with PBS, trypsinized with 0.05% trypsin/0.53 mM EDTA (Corning Cellgro), resuspended in PBS, pelleted at 1,500 rpm (maximum rotor radius: 20.78 cm) for 5 min at 4°C, and resuspended in 80% ethanol for 15 min on ice. The fixed cells were conjugated with Alexa Fluor® 488 azide to reveal HPG-labeling, and stained with NuclearMask™ Blue stain according to manufacture’s protocol (Invitrogen). The labeled cells were resuspended in PBS for flow cytometry analysis on a BD LSRII Multi-Laser Analyzer (BD Biosciences).
Nuclearmask blue stain
Manufactured by Thermo Fisher Scientific
NuclearMask™ Blue stain is a fluorescent dye used for staining and visualizing nuclei in biological samples. It binds specifically to DNA, enabling the identification and localization of cell nuclei in microscopy applications.
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Lab products found in correlation
2 protocols using nuclearmask blue stain
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Protein Synthesis Inhibition Assay
2
Measuring Protein Synthesis Inhibition
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To detect CHX-mediated inhibition of protein synthesis, Gli36-mCherry cells in a 24-well plate were prepared in parallel to the EV uptake and EV-RNA translation experiments as described above. At the indicated post-centrifugation time points, culture medium was replaced with methionine- and cysteine-free medium containing Click-iT HPG (Invitrogen) for 30 min, and the cells were washed with PBS, trypsinized with 0.05% trypsin/0.53 mM EDTA (Corning Cellgro), resuspended in PBS, pelleted at 1,500 r.p.m. (maximum rotor radius: 20.78 cm) for 5 min at 4 °C and resuspended in 80% ethanol for 15 min on ice. The fixed cells were conjugated with Alexa Fluor 488 azide to reveal HPG labelling, and stained with NuclearMask Blue stain according to the manufacturer's protocol (Invitrogen). The labelled cells were resuspended in PBS for flow cytometry analysis on a BD LSRII Multi-Laser Analyser (BD Biosciences).
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