Puromycin treatment in the presence of other translation inhibitors

Summary

This protocol for in vitro treatment of cells with puromycin is followed by immunofluorescence staining and fluorescence microscopy. Puromycin immunofluorescence allows quantification of the amount of nascent protein synthesis at a given point in time, but DOES NOT accurately report on the subcellular localization of nascent protein synthesis. See linked publication:

Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes

DOI: 10.7554/eLife.60048

Notes

For best imaging quality, cells should be cultured and fixed on a glass surface. This protocol assumes the cells are cultured in glass bottom well plates, but similar results can be achieved with glass coverslips inside standard plastic plates. Cells should be seeded 24–48 hr prior to treatment, and should be approximately 70–80% confluent at the time of experiments. Optimization of conditions for each cell line may be required. Cells in our experiments are grown in DMEM + 10% FBS, but the optimal media for growth of each specific cell line should be used throughout the experimental procedures. Note that we use emetine at 45 µM, as suggested by Bastide et al., 2018, which is ~4.6 times lower than the 208 µM used in David et al., 2012. Since maximal inhibition of translation is achieved at 1 µM emetine (Grollman, 1968), 45 µM emetine is thus a saturating concentration for inhibiting elongation. Note that when using pretreatment inhibitors, the same concentration should maintained throughout puromycin treatment and washing. Note also that magnesium should be added to the wash and fixation buffers to prevent ribosomal dissociation.

Materials

• Dulbecco’s modified eagle medium (DMEM, Life Technologies, catalog #11965118)
• 10% fetal bovine serum (FBS, Life Technologies, catalog #16000044)
• Anisomycin (Sigma, catalog #A9789)
• Cycloheximide (Sigma, catalog #C7698)
• Emetine dihydrochloride (Sigma, catalog number #E2375)
• Puromycin (Sigma, catalog #P7255)
• Phosphate-buffered saline (PBS)
• Magnesium chloride
• Sucrose
• Paraformaldehyde
• Tris-buffered saline (TBS)

Procedures

1. Prepare media aliquots supplemented with appropriate concentrations of elongation inhibitors, and pre-warm to 37°C. For inhibitor pretreatment, media should be prepared with inhibitors at 1X concentration (Cycloheximide at 355 µM, anisomycin at 37 µM, emetine at 45 µM). For puromycin treatment, media should be prepared with pre-treatment inhibitors at 1X concentration and with puromycin at 2X concentration (20µM in the media aliquot; 10µM final).
2. Remove cells from incubator and quickly remove media. Immediately add pre-warmed pretreatment media and return to incubator for 5 minutes.
3. Remove cells from incubator and quickly add an equal volume of puromycin treatment media to the cells. This media should contain 20 µM puromycin (will be 10µM final) and 1X concentration of pretreatment inhibitors (remains 1X). Return cells to incubator for 5 minutes.
4. Remove cells from the incubator and quickly remove media. Immediately place the plate on ice and quickly wash twice in ice-cold 1X PBS supplemented with 5 mM MgCl₂ and 1X concentration of pretreatment inhibitors.
5. After two washes, remove the PBS and fix cells by adding 1X PBS supplemented with 5 mM MgCl₂, 4% sucrose, and 4% paraformaldehyde (PFA). Incubate cells in fixative solution for 15 min at room temperature. 
6. Wash the cells three times with 1X TBS at room temperature.
7. After washing, proceed with immunofluorescence staining immediately, or store cells in TBS at 4°C for no longer than 24 hours before staining.