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.
The protocol was identical to that of the previous experiment, except for the use of a 561nm laser line to image the VEGF SmartFlares which are conjugated to Cy3 instead of Cy5 in the two control conditions. The VEGF and Scramble SFs were reconstituted on 09/03/2015 in nuclease-free water.
Uptake Control (Cy5)
As we saw previously, the HeLa cells appear to constitutively take up the FITC-dextran whereas only a subset of cells take up the Cy5-Uptake Control SmartFlare. Also the relatively punctate signal from both the FITC-dextran and the Cy5-Uptake Control SmartFlare again suggests that they are likely contained within endosomes.
VEGF SmartFlare (Cy3)
The signal from the Cy3-VEGF SmartFlare also appears punctate, similar to the Cy5-Uptake Control SmartFlare and the FITC-dextran. As with the Uptake Control (and unlike the dextran), some cells appeared to have more SmartFlare than others.
Scramble Control SmartFlare (Cy5)
Here we observe again, a punctate signal similar to the Cy5-Uptake Control. This is unexpected as this SmartFlare is sold as a negative control, that is used to estimate the background signal.
Authors: Gemma Carolan & Dave Mason