Transfection of Axol human Neural Progenitor Cells using Axol ReadyFect
Axol ReadyFectTM Technology Overview
Axol ReadyFectTM is based on the Tee-Technology (“Triggered Endosomal Escape”) that combines and exploits the properties of cationic lipids and polymers to achieve an extremely efficient DNA delivery into Axol human Neural Progenitor Cells . This powerful transfection reagent optimised to transfect Axol human Neural Progenitor Cells with improved cytoplasmic release and better biodegradability without impacting phenotype and differentiation potential.
The instructions given below have been validated on Axol human Neural Progenitor Cells . We recommend to use 3 µL of Axol ReadyFectTM / 1 µg of DNA. You can use your complete Axol Neural Maintenance medium, except during the preparation of the Axol ReadyFectTM / DNA complexes where you should use PBS.
Nucleic acids should be as pure as possible. Use transfection grade plasmid DNA (high degree of supercoiled forms) to achieve high expression. Avoid long incubation time of the DNA solution in PBS before the addition of Axol ReadyFectTM to circumvent any degradation or surface adsorption.
Considerations Before You Begin
1. Thaw and culture your Axol human Neural Progenitor Cells according to our instruction manuals.
2. Ensure that the cells are 50-70% confluent at the point of transfection.
3. DNA and Axol ReadyFectTM solutions should be at room temperature and be gently vortexed prior to use.
4. Calculate the amount of Axol ReadyFectTM that you will require. Use 3 μL of Axol ReadyFectTM per μg of DNA, as this is the optimal ratio (3:1) for Axol hNPCs. However, depending on your experimental requirements, some optimization may be required. In this case, please follow suggestions in the Optimization section of this manual.
Warning: Do not let your hNPC culture exceed 70% confluence as this will reduce your transfection efficiency. In the representative images below, 50-60% confluence is ideal whereas 80% confluence is too high.
Standard Transfection Protocol
1. Prepare your DNA solution by diluting 0.125 µg to 10 µg of DNA in 25 µL to 350 µL of PBS according to guidelines in Table 1.
3. Add DNA solution to the Axol ReadyFectTM solution, mix gently by vortexing slowly or pipetting up and down 4-5 times.
4. Incubate the mixture for 20 mins at room temperature.
5. Add the mixture to Axol hNPCs (growing in their culture medium) dropwise and homogenize by gently rocking the plate side to side to ensure a uniform distribution of the mixture.
6. Incubate the cells at 37°C, 5% CO2.
7. Evaluate your transgene expression 24 to 72 hrs post-transfection.