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.
- HeLa cells were seeded at 500,000 cells per well of a 35mm glass-bottomed dish.
- While the cells settled, stock solutions were made up for FITC-dextran and the Cy5-Uptake Control SmartFlare. The FITC-dextran was prepared to a concentration of 10 mg/mL in PBS and the Cy5-Uptake SmartFlare was reconstituted into 1 mL of nuclease free water.
- The manufacturers of the SmartFlares recommended to add 40μL to a 2 mL volume, so 20 μL of the Cy5-Uptake Control SmartFlare was added to 100 μL FITC-dextran (to give a final concentration of 1 mg/mL) and 880 μL MEM medium, which also contains Fetal Bovine Serum (FBS), non-essential amino acids and 1% penicillin and streptomycin.
- Existing medium was removed from the settled cells and a 2 mL PBS wash was carried out twice. Once all PBS was removed 1 mL of the SmartFlare/dextran/media mixture was added to the cells. The cells were then placed back into a 37°C, 5% CO2 incubator for 18 hours.
- Before imaging, all liquid was removed from the cells and another 2 mL PBS wash was carried out 3 times. 1 mL of fresh medium was then added back onto the cells for imaging.
- Cells were imaged live (at 37°C, 5% CO2) on the Zeiss Multiphoton LSM510 using a 63x (Plan-Apochromat 1.4NA oil) objective. The 488nm and 633nm lasers were used to image the FITC-dextran and Smartflare-Cy5 (uptake control) respectively. The scan mode was set to 1024 x 1024 using a 4x averaging line scan. There was also a transmitted-light image captured on the 488nm line.
We’re still trying to figure out the best way to provide the raw data. For the time being you can find some example data from this experiment here.
UPDATE: You can now browse all of the project data at our Open Data Repository.
1st Observation: HeLa cells constitutively endocytose fluid phase markers (do I hear Sweden on the line?).
After imaging around the dish it appeared that the majority of the HeLa cells had taken up the FITC-dextran showing that they are carrying out constitutive endocytosis. The relatively punctuate signals also suggest that that dextran is contained within endosomes.
2nd Observation: SmartFlare uptake varies between cells.
HeLa cells loaded with SmartFlare Uptake Control for 18h. Showing the full dynamic range of intensity (1-4096)
And the overlay of the two images above:
Unlike the FITC-dextran the Cy5-Uptake SmartFlare doesn’t appear to be taken up by all the cells. To get a better view of the distribution of the SmartFlare, the dynamic range of the intensity was altered (below) to make the dimmer signals more obvious.
When the channel is scaled up it is possible to see that some more cells do take up the Cy5-Uptake SmartFlare but at lower amounts.
3rd Observation: SmartFlare signal appears largely punctate in cells.
Cropping into a cell shows that there is both punctate and in some cases, diffuse signal.
Another example is below.
The signal from the SmartFlares largely seems to be punctuate, as with the FITC-dextran this would suggest that they too are contained in endosomes.
Authors: Gemma Carolan & Dave Mason