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.
The idea is to treat the cells with SmartFlares (+/- DMOG to stimulate VEGF transcription), then fix and immunostain for markers of various cellular compartments and organelles. The images can then be analysed for co-localisation of the probes. If certain probes co-localise, it can be used as an indication that the SFs are present within those compartments.
There are two main caveats to this idea:
- There is no precedence for the fixation of the SFs
- You can only make conclusions about the probes that you stain for
In terms of fixation, we typically use paraformaldehyde (PFA) to covalently cross-link primary amines. Whilst this would have no effect on the gold, it should cross-link DNA and RNA oligonucleotides, thus preserving the location of the SFs.
For the second choice (and given the continued punctate nature of the SmartFlares) it seems worthwhile to investigate various aspects of the endocytic pathway. Given an educated guess about where the SmartFlares are located, we decided to probe for markers of the terminal lytic compartments; the lysosomes (LAMP1) and the recycling compartments (typically localised using the Transferrin Receptor). We also decided use a cis-golgi marker (GM-130) as a negative indicator, as the SmartFlare shouldn’t be localised here. Because the Uptake/Scrambled Controls and VEGF SmartFlares are Cy5 and Cy3-labelled respectively, anti-Rabbit Alexa555 and anti-Rabbit Alexa647 secondary antibodies will be used. All three of the SFs will be treated with and without DMOG therefore, a single replicate of the experiment will look like this (a more detailed protocol follows):
- HeLa cells were seeded at 400,000 cells /well
- 3 mL of each of the SmartFlares were prepared with FITC-dextran, MEM medium (which also contains Fetal Bovine Serum (FBS), non-essential amino acids and 1% penicillin and streptomycin) +/- DMOG as below.
- 300 μL FITC-dextran, 60 μL of SmartFlare and 2640 μL MEM medium
- 300 μL FITC-dextran, 60 μL of SmartFlare, 15 μL DMOG and 2625 μL MEM medium
- 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.
- Medium removed from the cells and a 2 mL PBS wash carried out 3 times
- 1.2 mL of 4% PFA was added to each of the dishes and left on for 30 minutes at room temperature
- PFA removed after the 30 minutes and another 3 times 2 mL PBS wash was carried out
- 2 mL of PBS was left on the cells and they were stored at 4°C
- During all waiting periods the cells were kept under foil to protect from light
- Cells were permeabilised and blocked by adding 0.1% TritonX-100: 1% BSA:PBS for 60 minutes at room temperature
- While this occured, Primary antibodies were prepared in blocking buffer: 1:500 dilution, so 2.4 μL of each antibody was separately added to 1200 μL 1% BSA:PBS
- After 60 minutes the 0.1% TritonX-100: 1% BSA:PBS was removed and the cells washed 3 times with 2 mL PBS
- 200 μL of each primary antibody was added to the appropriate dish for 75 minutes at room temperature
- Secondary antibodies were prepared in blocking buffer using a 1:1000 dilution: 1.2 μL of each antibody separately added to 1200 μL of 1% BSA:PBS
- After the incubation the antibody is washed off with another 3 times 2 mL PBS wash
- 2 mL of PBS added to the cells and stored in the fridge until imaging
- Cells were imaged on the Zeiss Multiphoton LSM510 using a 63x (Plan-Apochromat 1.4NA oil) objective. The 488nm, 561nm and 633nm lasers were used to image the FITC-dextran, VEGF SmartFlares and control SmartFlares 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.
Authors: Gemma Carolan & Dave Mason