This week I have been evaluating osmotic lysis of pinocytic vesicles as a technique for cellular delivery. In the preliminary stages of my project I will be loading dextran into the cells.
Protocol used has been adapted from the following papers;
Tracking Individual Intracellular Proteins Using Quantum Dots
Introduction of macromolecules into cultured mammalian cells by osmotic lysis of pinocytic vesicles
Protocol
HeLa cells were seeded into a 6W dish (2 ml/well) containing cover slips at a cell density of 4×105 cells/well aiming for 50-60% confluence and were left for 24 hours to adhere.
The culture medium was then replaced with 0.5 ml hypertonic media (F12-HEPES + 0.5M sucrose + 10% PEG 1000 + GNPs) with dextran (0.1 mg/ml dextran) and incubated for 10-30 minutes. As a control, cells were treated with only hypertonic media. Media was then switched to hypotonic media (6:4 F12:dH20) for 2 minutes then back into regular F12. Cells were then left for up to 1 hour to recover.
The cells were fixed in 4% PFA for 10min at RT. Cells were then washed twice with PBS (the second including 1:100 DAPI) and mounted for imaging.
Cells will be imaged using an epifluorescent microscope. x20 objective lens will be used to image 20 fields in each sample. x63 will be used to image individual cells for punctate or diffuse signal.
Anticipated outcome
When imaging the cells I expect to either visualise dextran in the cells, indicating that the osmotic lysis was successful or see no fluorescence indicating that dextran has not been delivered into the cells. In the control cells I also expect to see no fluorescence. Furthermore of interest will be if the signal is diffuse or punctate.
If no fluorescence is seen in the dextran loaded cells the experimental design will be altered. If there are issues I expect it will either be with dextran concentration or treatment time.
Rapha-z-lab-commons 