Troubleshooting & Optimization

Problem	Solution
No / few cells have Cbl-fluorophore probe.	Optimize bead loading by varying cell density, probe concentration, mechanical loading step. Use a fluorescent dye that is not cell permeable to optimize the procedure.
Fluorescence signal from Cbl-fluorophore probe is dim.	Increase Cbl-fluorophore probe concentration (Table 3) and/or volume of stock added to cells (up to 20 μL per imaging dish).
No / few cells have NLS-TagBFP transfection marker.	Optimize transfection efficiency of U-2 OS cells by varying cell density.
Riboglow recruitment to stress granules results in very high fluorescence ratio cytosol/granule (>15).	Signal is likely too dim and close to the background, select cells with higher probe concentrations.
No mRNA at stress granules / no U-bodies detected.	Verify production of Riboglow-tagged RNA and colocalization with stress granules / U-bodies by fluorescence in situ hybridization (FISH). See (Braselmann et al., 2018) for protocols and probe sequences. Verify transfection of tagged RNA by producing reporter gene (fluorescent protein) from the same plasmid.
No cells with cytosolic granules detected in U-body assay.	Verify transfection by repeating with a transfection marker plasmid encoding for a fluorescent protein, determine transfection efficiency (should be >80% for HeLa cells).
Cytosolic granules detected in both experimental and control condition for U-body assay.	Reduce Cbl-fluorophore probe concentration to avoid non-specific probe aggregation. A control without Thapsigargin-treatment should not result in granule or aggregate formation.