Got a query? Check out our frequently asked questions.
How do you store CPNs™ and what is their shelf life?
CPNs™ can be stored under normal conditions at room/ambient temperatures and have a shelf life of 24 months. Once conjugated to biological material, it is recommended to maintain the storage temperature at 2°C to 4°C.
How thermally stable are CPNs™?
CPNs™ have been tested up to 120°C in an autoclave and are extremely stable. In fact, CPNs™ demonstrate a very slight increase in fluorescence at 120°C. Therefore, there is the potential to use the CPNs™ in a thermocycler and anneal nucleotides to target sequences linked to the CPNs™.
Can CPNs™ be frozen?
CPNs™ can be frozen without loss of fluorescence in standard aqueous buffers. Cell samples and tissues that have been frozen can also be labelled with CPNs™.
Does a change in pH have an effect on CPNs™?
CPNs™ have been tested from pH 4 to pH 10 with no detrimental effects on fluorescence.
Can antibodies and other moieties be attached to CPNs™ by means other than steptavidin and biotinylated conjugation?
Yes, the surface chemistry of CPNs™ allows direct conjugation via free carboxylic acid groups using N-ethyl-N’-dimethylaminopropyl-carboiimide (EDC) chemistry. A conjugation protocol is included on each of the product sheets.
Are CPNs™ cytotoxic?
The Component parts of CPNs™ are non-toxic and the CPNs™ themselves have demonstrated no signs of toxicity after cell loading, and over 6 days in an IncuCyte®.
What determines the excitation and emission of wavelengths of the CPNs™?
It is the chemical structure of the polymer at the core of the CPNs™ that determines the wavelengths, allowing us to ultimately develop specific CPNs™ to cover the visible and infrared spectrum.
What are the excited state lifetimes?
The excited state lifetimes for CPNs™ are in the nanosecond range and have been measured at 5ns for CPN™ 680 and 0.7ns for CPN™ 550.
Can a centrifuge be used for purification as an alternative to magnetic pull down?
CPNs™ have been tested in a centrifuge at 10,000xg for 5 minutes after which the CPNs™ readily resuspend and continue to fluoresce as expected.
Can the CPNs™ be concentrated using centrifugation?
The CPNs™ have been successfully pelleted by centrifugation at 10,000 xg for 5 minutes with no change in fluorescence or size parameters. Care should be taken once the CPNs™ are linked to proteins or other molecules. This is due to some proteins, including streptavidin and antibodies, having been shown to promote aggregation of CPNs™ upon centrifugation.
Can CPNs™ be purified with a spin filter?
We have exchanged purified, exchanged buffers and concentrated CPNs™ using spin filtration columns. The CPNs™ work optimally with cellulose acetate membranes but can be used with polyethersulfone (PES) membranes if they are suitably passivated (see protocol link).
Can Tween be used with CPNs™?
CPNs™ can be used with Tween20 or Triton-X-100 (up to 1% of each shows no detrimental effects on the CPNs™ performance or fluorescence, higher concentrations have not been tested).
Can CPNs™ be used in applications other than in-vitro R&D?
Yes. CPNs™ are a true platform technology with a multitude of different applications extending beyond in-vitro R&D labelling and tracking applications. CPNs™ can be developed for use in disease diagnostics, bio-hazard detection in food and water hygiene (cryptosporidium is an ongoing problem for water utilities) and therapeutics, i.e. the imaging of tumours with infra-red CPNs™ and MRI (multi-modal). Outside the ‘normal’ life sciences CPNs™ can be used in agri-tech in the form of field-based diagnostics to improve plant and crop disease diagnosis and within forensics with the potential to provide ‘on site’ crime scene identification of biological samples, and magnetic collection of samples.
Can CPNs™ be freeze dried?
CPNs™ have been successfully freeze dried and reconstituted without it affecting their performance.
Do CPNs™ sediment if stored for a long time?
If left for over 4 weeks then slight sedimentation of the CPNs™ may occur. The CPNs™ can be readily resuspended with brief agitation or vortex mixing. Sedimentation is not due to aggregation and the CPNs™ remain as mono-dispersed particles.
Can CPNs™ be sterilised for cell culture and in vivo applications?
CPNs™ are typically filter sterilised using a 0.22μm filter. CPNs™ can also be autoclaved in suspension with no detectible effects on their performance.
Once linked to my protein how should I store the CPNs™?
The protein linked CPNs™ will be susceptible to microbial growth. It is therefore recommended that sodium azide (3.2mM/0.002% w/v) is added to the suspension, with it being stored at 4°C. The CPN™ part of the linked nanoparticle will continue to remain stable, so any optimal storage conditions for your protein of interest should be applied when storing the linked nanoparticles.
How do you load CPNs™ into cells via the endocytic pathway?
Cells are loaded with the CPNs™ using an overnight incubation (16-24 hours). The CPN™ solution (0.1mg/ml) is diluted 1 in 10-100 in the standard culture media. Typically, 5-50 CPNs™ are taken up per cell, however the amount of CPN™ taken up varies between cell types and depends on how the cells are growing.
CPNs™ are taken up by endocytosis and trafficked through the endosomal/lysosomal pathway. This is clearly visible as bright, perinuclear puncta when imaged using fluorescent microscopy.