Direct Microinjection of SmartFlares

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.

Microinjection
Image Courtesy of the Liverpool Centre for Cell Imaging

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Imaging SmartFlares +/- DMOG (Live)

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.

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Imaging the localisation of SmartFlares – experimental design

As discussed in a previous post, the punctate distribution of all of the SmartFlares (SFs) is both unexpected and curious. In order to better understand where the SFs are localised in the cell, we would like to do a fairly routine co-staining experiment.

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Imaging of VEGF, Uptake Control and Scramble SmartFlares

Despite the reported stability of SmartFlares (which according to the brochure is up to a year at room temperature after reconstitution), we have until now, not opened and reconstituted the VEGF and Scrambled SFs, wanting to make sure that our biological system was in place before we did so.

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