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.

Protocol

Results

All of the data acquired are available at our Open Data Repository date stamped [2015-04-15]. The following labelling scheme is used:

2015-04-15_scheme

VEGF SmartFlare (Condition F)

2015-04-15-F1

Control SmartFlares (Conditions D & E)

First the Uptake Control SmartFlare:

2015-04-15-D1

And the Scrambled Control SmartFlare:

2015-04-15-E1

Effect of DMOG Treatment

First looking at the VEGF SmartFlare:

2015-04-15-C_F

Then the Uptake Control SmartFlare

2015-04-15-A_D

And the Scrambled Control SmartFlare:

2015-04-15-B_E

Summary

  • All of the cells observed, took up dextran which had a punctate distribution in the cells.
  • Without DMOG, signal was observed in the VEGF SmartFlare condition (using Cy3 optics) as well as both of the Control conditions (using Cy5 optics). In all cases the signal was punctate.
  • In each of the SmartFlare conditions the signal was visible in a subset of cells positive for dextran. The SF containing compartments were not exclusively dextran positive (quantitation to come).
  • DMOG treatment (qualitatively) did not seem to alter the intensity or localisation of SmartFlares.
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