This week I have been evaluating scrape-loading 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; Scrape-loading and dye transfer. A rapid and simple technique to study gap junctional intercellular communication. Gold nanoparticle-mediated (GNOME) laser perforation: a new method for a high-throughput analysis of gap junction intercellular coupling. Dipyridamole increases gap junction coupling in bovine GM-7373 aortic endothelial cells by a cAMP-protein kinase A dependent pathway. A method for incorporating macromolecules into adherent cells.
HeLa cells were seeded into a 6W dish (2 ml/well) 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 100uL of 1 mg/ml TMR-dextran (10kDa) in a bath solution of PBS and the monodispersed cells were scraped at room temperature using a rubber policeman. As a control, cells were scraped without dextran.
Cells were then centrifuged at 500x g for 3 minutes to a pellet and resuspended in growth media. The cells were then seeded onto coverslips in a fresh 6W dish.
The cells were left for 3.5 hours to adhere 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 fields, 20 fields in each sample with be imaged. x63 objective will also be used to image. DIC, DAPI and DsRed will be used.
When imaging the cells I expect to either visualise dextran in the cells, indicating that the scrape-loading 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.
If no fluorescence is seen in the dextran scrape-loaded cells the experimental design will be altered. One area which could be explored is adding components to the bath solution in which the cells were scraped to increase cell viability, these include; +/- Glucose, +/- Ca2+/Mg2+.
Further issues may have been with the incubation time after the cells were loaded, the cells may not have been monodispersed, dextran concentration may have been too high or there may have been issues with cell density.