The hTERT RPE-1 cells and NGLY1−/−, PMM2F119L/−, and DPAGT1+/− mutant lines were plated in 384 well plates at a density of 3000 cells per well. The next day compounds were added to the cells at a final concentration of 10 µM and incubated for 24 h. Cells were stained with MitoTracker Red (Molecular Probes, cat. M7512) mitochondrial stain for 30 min according to the manufacturer’s protocol, then media was removed, cells were fixed in 4% PFA/PBS, permeabilized with 0.1% NP-40, and blocked with 3% BSA/ PBS overnight. For staining, ConcanavalinA-488 (Molecular Probes, cat. C11252), phalloidin-547 (Molecular Probes, cat. A22283), and DAPI were added to the wells, then washed before imaging. Images were acquired on a Molecular Devices Image Express microscope at 4 fields per well. Feature extraction from images was done with Perkin-Elmer Columbus Image Analysis software, and feature analysis and hit determination was performed using TIBCO Spotfire analysis package.
Concanavalina 488
Manufactured by Thermo Fisher Scientific
ConcanavalinA-488 is a fluorescent conjugate of the lectin Concanavalin A, which binds to carbohydrates containing alpha-mannopyranosyl and alpha-glucopyranosyl residues. It is used as a cell surface marker and for the detection of glycoproteins.
Automatically generated - may contain errors
2 protocols using concanavalina 488
1
Multiparametric Imaging of Organelle Morphology
2
High-Throughput Imaging of Mitochondrial Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The hTERT RPE-1 cells and NGLY1 -/-, PMM2 F119L/-, and DPAGT1 +/-mutant lines were plated in 384 well plates at a density of 3,000 cells per well. The next day compounds were added to the cells at a final concentration of 10 µM and incubated for 24 hours. MitoTracker Red (Molecular Probes, cat. M7512) mitochondrial stain was added to the media, following the manufacturer's protocol. After 30 minutes of labeling, media was removed, cells were fixed in 4% PFA in PBS, washed, permeabilized with 0.1% NP-40, and blocked with 3% BSA in PBS overnight. For staining, ConcanavalinA-488 (Molecular Probes, cat. C11252), phalloidin-547 (Molecular Probes, cat. A22283), and DAPI were added to the wells, then washed before imaging. Images were acquired on a Molecular Devices Image Express microscope at 4 fields per well. Feature extraction from images was done with Perkin-Elmer Columbus Image Analysis software, and feature analysis and hit determination was performed using TIBCO Spotfire analysis package.
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