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.
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.
As mentioned before, one of the things that has been bugging me about the SmartFlares is that they’re not where anyone expects them. EMD expect them in the cytosol (we don’t see them there), I expected them in endosomes and they weren’t really there either. So what could this mystery compartment be?
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 a recent post, I was musing about why the SmartFlares weren’t in the swollen chloroquine-induced vacuoles. Out of interest more than anything, I did a quick experiment to see what would happen if I pre-labelled the endocytic pathway with one coloured dextran, then added in a second with the chloroquine.
I recently discussed the SmartFlare project at a joint Sée and Lévy Group Meeting, partially extolling the virtues of Open Science but mostly focussing on the data acquired so far and some of the proposed experiments.
Regarding the SmartFlare project, the three big questions that formed the basis of the talk were:
- Why do only some cells take up SmartFlares?
- Where are the SmartFlares?
- Why are the VEGF and Scrambled SmartFlares fluorescent in puncta?
Below are some of the interesting points that were made during the meeting. Replies at the time (as best as I can remember them) in green: