- What does the product look like?
- How should I reconstitute? The fluorophore is red, but the solution is blue - have I made it up properly?
- What excitation and emission wavelengths and filter sets should be used?
- Cells don’t bind the fluorescent ligand
- Cells label everywhere (cytoplasm, nucleus), not just the membrane
- Control (non target-expressing) cells label as well as the target cells
- There is very high background fluorescence
- Cells label well, and show a good saturation-binding curve – but the maximal saturation binding level is lower than expected
- There are bright fluorescent particles in the cell cultures
The products are supplied as a dried film of blue material adhering to the inside of the conical bottom of a ‘champagne flute’ brown glass vial. The product should be visible as a dark smear through the side of the vial.
2. How should I reconstitute the fluorescent ligand? The fluorophore is red, but the solution is blue - have I made it up properly?
Check the individual datasheet for reconstitution details. Generally, we recommend the following:
If dissolved in DMSO at 10mM according to the instructions in the datasheet, the product will appear to be a dark blue solution.
For sub-aliquoting in slightly larger volumes, the product can also be reconstituted at 1 mM by adding 10x the volume of DMSO stated.
The product can be further diluted in DMSO, for example to 100 μM, and will retain its blue colour.
Final dilution to ~ 100 nM is in buffer (+ 0.1% DMSO)
For emission, use a narrow-band filter with a peak at 650 nm.
Shorter wavelength excitation and longer wavelength emission filters can be used but signals will be weaker, and there may be cross-talk with other fluorophores.
Each fluorescent binding ligand product is provided with a datasheet that recommends the appropriate excitation wavelength and emission filter-set for optimal fluorescence detection of the product.
- Cells are not expressing the receptor
- Compare with cells from another source
- Compare with cells at different growth stages
- Use the ligand for FACS to select cells expressing the receptor
- The receptor is not reaching the cell membrane
- Ligands do not readily cross the cell membrane, so this is difficult to test
- An antibody approach with fixed, permeabilised cells might confirm this
- The receptor is unable to bind the ligand.
- Fixing the cells can prevent ligand binding – use live cells
- Too much solvent (DMSO) in buffers can damage the cells
This is likely to be because the cells are not healthy and have taken up the ligand non-specifically
- Check the culture conditions – cells should not be over-confluent
- Check the assay conditions – pH should be 7.4 in isotonic buffer (e.g. HBS or PBS)
- Ensure that minimal solvent is used (e.g. DMSO < 0.1%)
- Check the cells have not been fixed and permeabilized
Even in a healthy culture, there will be dead cells that show non-specific staining
- Make sure cell-plates are handled carefully and removed from the incubator immediately before use, not left sitting on the bench for long periods
Likely cause: high non-specific binding to control and target cells
- Ensure that both control and target cell lines are healthy (see previous FAQ)
- Ensure the ligand is not being used at too high concentration. (The maximu should be 100nM)
- Check if the ligand is binding to an endogenous receptor expressed by the host cells by blocking with an appropriate unlabelled competitor
- Abcam provides some cross-selectivity data, but the ligands may bind to other receptors
- Use the pharmacology of the unlabelled drug from which the ligand is derived as a guide to potential additional targets to which the ligand may be binding
- Test this with selective unlabelled blockers for the off-target receptor
Cross-reaction to other receptors
BODIPY-630/650 should not show this because it’s fluorescence is quenched in aqueous solution. If this is reported, it is likely to be due to a high concentration of the ligand in solution around the cells.
- Use the ligand at a lower final concentration (30 – 100nM gives the best signal : background)
- Add a brief (10 min), gentle wash step to reduce the background fluorescence
8. Cells label well, and show a good saturation-binding curve but the maximal saturation binding level is lower than expected
This may be due to the fluorescence detector reaching saturation, rather than the cells reaching binding saturation.
- Try reducing the gain on the detector
- Try taking measurements on an alternative instrument
Explanation: microscopic crystals of ligand have precipitated out of solution onto the cells.
Likely cause: the ligand is not completely dissolved in DMSO before use.
- After thawing the DMSO stock aliquot of ligand, sonicate it briefly to ensure the ligand is dissolved and the solution is homogeneous
- Then, dilute the ligand to 2x final concentration in buffer for addition to an equal volume of assay buffer covering the cells
- Do not repeatedly freeze/thaw the stock ligand aliquots – it can cause crystals to form
- Do not freeze/thaw the final ligand-buffer solution – this can also cause crystals to form