Table 2.
Troubleshooting Guide for Detection of LC3‐Associated Phagocytosis
| Problem | Solution |
|---|---|
| Basic Protocol 1 : Detection of LAP by electron microscopy | |
| Holes in membranes | Begin dehydration series with lower concentration of ethanol |
| Cell structures look small | Use alternative fixative |
| Adjust concentration of salt to avoid hypertonic solution | |
| Basic Protocol 2 : Detection of LAP by confocal microscopy of LC3‐GFP‐expressing cells | |
| Alternate Protocol 1 : Detection of LAP by confocal microscopy using immunofluorescence | |
| Not many cells in chamber slide | Use different extracellular matrix solution or allow cells to adhere longer |
| Excessive LC3‐GFP puncta | Increase percentage of serum in cell culture medium |
| Speckled LC3‐GFP | Reduce dilution of anti‐LC3 antibody |
| Basic Protocol 3 : Detection of LAP using flow cytometry of LC3‐GFP‐expressing cells | |
| Alternate Protocol 2 : Detection of LAP using antibody staining and flow cytometry | |
| No difference in LC3‐GFP fluorescence in positive control or other samples | Increase concentration of or incubation with digitonin solution |
| Increase rapamycin concentration or incubation time | |
| Excessive LC3‐GFP fluorescence in negative control or other samples | Decrease concentration of or incubation with digitonin solution |
| Basic Protocol 4 : Detection of LAP by western blot of purified LAPosomes | |
| No bands or signal | Increase cell number for harvest |
| No UNC93B bands in sample | Repeat purification to avoid contamination with proteins not from bead‐containing phagosomes |