Imaging Techniques Cell Biology

scientificprotocols authored about 3 years ago

Authors: Jean-Philippe Pellois

Abstract

We report that a tetramethylrhodamine-labeled dimer of the cell-penetrating peptide TAT, dfTAT, penetrates live cells by escaping from endosomes with high efficiency. By mediating endosomal leakage, dfTATalso delivers proteins into cultured cells after a simple co-incubation procedure. We achieved cytosolic delivery in several cell lines and primary cells and observed that only a relatively small amount of material remained trapped inside endosomes. Delivery did not require a binding interaction between dfTATand a protein, multiple molecules could be delivered simultaneously, and delivery could be repeated. dfTAT-mediated delivery did not noticeably affect cell viability, cell proliferation or gene expression. dfTAT-based intracellular delivery should be useful for cell-based assays, cellular imaging applications and the ex vivo manipulation of cells.

Materials

  1. Heparin (Sigma, CAS 9041-08-1).
  2. SYTOX® Blue (Invitrogen, S11348) AND SYTOX® Green (Invitrogen, S7020)
  3. nrL15 L-15 (–) cysteine(Hyclone, special order). L-15 can also be used despite containing Cysteine. Cysteine can in principle reduce dfTAT to the dramatically less active monomer fTAT. However, cysteine oxidizes in L-15 to yield cystine. dfTAT is therefore relatively stable in L-15. However, nrL-15 is recommend to eliminate possible experimental variability.
  4. dfTAT: synthesis protocols are described in “REF”. Samples will be provided upon request (contact: [email protected]). To date, the chemical features important for the endosomolytic activity of dfTAT are not fully understood. Simple changes in the structure of dfTAT might therefore reduce its activity.

Equipment

  1. Biosafety cabinet
  2. Inverted epifluorescence microscope (Model IX81, Olympus) equipped with a heating stage maintained at 37 °C and with a Rolera-MGI Plus back-illuminated electron-multiplying charge-coupled device (EMCCD) camera (Qimaging).
  3. 37 °C humidified, 5% CO2 incubator

Procedure

  1. Seed cells (HeLa, HDF, etc) in a dish (e.g. 8 well or 24 well dish).
  2. Grow cells in appropriate medium (e.g. DMEM supplemented with 10% FBS and Pen/Strep) until 80-90% confluency in a 37°C humidified atmosphere containing 5% CO2.
  3. Wash cells 3 times (3X) with PBS.
  4. Make a 5 µM working concentration of dfTAT by diluting dfTAT in nrL-15 media (for a 8 well dish the total volume should be 200 µL). A concentration of 5 µM dfTAT leads to efficient delivery (high level of cytosolic delivery in more than 90% of cells present in a dish) in most cell types tested to date. However, lower or higher concentrations might be more adequate for cell types with a high or low propensity for penetration.
  5. Incubate cells with dfTAT with or without cargo (e.g. EGFP, TAT-Cre etc.) and keep at 37°C for 1h (incubation time can be reduced but dfTAT typically requires approximately 30 to 45 min to induce endosomal leakage).
  6. Wash cells with heparin (1 mg/ml) in L-15 (3 washes are recommended) to remove dfTAT bound to the plasma membrane of cells.
  7. Incubate cells with cell-impermeable nuclear stain such as Sytox Blue or Sytox Green (2 µM in nrL-15) to determine whether the plasma membrane of cells is compromised (dead cells will be stained while live cells will not).
  8. Image cells using a fluorescence microscope (100X oil immersion or 20X objective). dfTAT is imaged using a RFP filter (Ex = 560 ± 20 nm/Em = 630 ± 35 nm). Successful delivery leads to a diffuse fluorescence of dfTAT throughout the cell (assessing the delivery of protein or peptide of interest will depend on application). Staining of nucleoli by fTAT (the reduced product of dfTAT upon cytosolic entry) can be used as an indication that the fluorescence detected is intracellular. fTAT will degrade within few hours. At this point, the fluorescence of the degradation fragments will appear as punctate. This should not be confused for the punctate distribution that is also seen if dfTAT remains unsuccessfully trapped inside endosomes (this can happen if dfTAT is present at too low of a concentration).

Troubleshooting

  1. Cells should not be overly confluent (> 90% confluency) since this might affect the delivery efficiency. Cells should also be healthy: dead cells in the culture can release apoptotic fragments with which dfTAT can interact (e.g. DNA from degraded nuclei). This in turn can interfere with delivery efficiency and the quality of imaging.
  2. Cells should be washed thoroughly, especially removing FBS before adding dfTAT. BSA present in FBS can bind to dfTAT and this can lower the delivery efficiency of dfTAT. Washes should however be performed with care as excessive force might cause adherent cells to detach from the culture dish.
  3. When delivering a protein/ peptide using dfTAT, the protein/peptide stock solution sample should be sufficiently concentrated to avoid excessive dilution of the nrL-15 media during incubation: e.g. adding on 5-10 µL of sample to 200 µL nrL-15 is recommended.

References

Erazo-Oliveras, A. et al. Protein delivery into live cells by incubation with an endosomolytic agent. Nature methods 11, 861-867 (2014).

Source: Protocol Exchange. Originally published online 8 September 2014

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