Throughout all of the fluorescent microscopy experiments, we’ve been seeing SmartFlare fluorescence in punctate structures. Using these techniques, it’s very difficult to say with certainty that the SmartFlares are within vesicles or (for example) aggregated/agglomerated in the cytosol. As this is pertinent to the current investigation, we took cells loaded with SmartFlares to the electron microscope which has the resolution to discern membrane structures as well as the gold particles.
We’ve so far, not seen any response of the VEGF SmartFlares to treatment of our cells with DMOG. Combined with the punctate distribution of the signal, we have no evidence that the SmartFlares make it to the cytosol in 18 hours. As we’re not sure if the problem lies in the SmartFlares or the delivery, I microinjected the SmartFlares directly into the cells thus bypassing the need for endosomal uptake.
In order to investigate why the SmartFlares seem to be fluorescent in puncta within the cells, we designed an experiment to try to inhibit endosomal maturation, thus (hopefully) preventing SmartFlare processing and release of fluorescence.
The Uptake Control SmartFlare was opened first to help us to establish a loading protocol. As such, the physical characterisation of that SmartFlare was performed in an earlier post. Thanks once again go to Joan Comenge for taking the VEGF and Scrambled control SmartFlare to the Transmission Electron Microscope (TEM).
This is a final repeat of the basic SmartFlare experiment. HeLa cells are loaded with the three variants of SmartFlares and imaged 18h later.
The purpose of these experiments is to be able to visualise the SmartFlares (SFs) in the cell after having followed the loading protocol provided by the manufacturers. After 16-20h the SFs should be in a position to interact with mRNA in the cell and this means a cytosolic localisation. We will use DMOG to stimulate VEGF mRNA production in the cell.