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Transfection of Axol Neural Stem Cells with ReadyFect Protocol

Product Information

Product Information

Axol ReadyFect TM Technology Overview

Axol ReadyFect TM 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 (hNPCs).

Axol ReadyFect TM is a powerful reagent optimised to transfect Axol hNPCs with improved cytoplasmic release and better biodegradability without impacting phenotype and differentiation potential.

The instructions given below have been validated on Axol hNPCs. We recommend to use 3 μL of Axol ReadyFect TM / 1 μg of DNA. You can use your complete Axol Neural Maintenance medium, except during the preparation of the Axol ReadyFect TM / 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 ReadyFect TM to circumvent any degradation or surface adsorption.

Axol ReadyFect TM leads to 30-40% transfection efficiency. The image demonstrates transfection results using 3 μL of Axol ReadyFect TM / 1 μg of a GFP-encoding vector (pVectOZ-GFP). GFP expression was evaluated 2 days post-transfection.

Considerations Before You Begin

  1. Thaw and culture your Axol hNPCs or Axol AD hNPCs according to our instruction manuals, which can be found at axolbio.com/instructioon-namuals/s.
  2. Ensure that the cells are 50-70% confluent at the point of transfection.
  3. DNA and Axol ReadyFect TM solutions should be at room temperature and be gently vortexed prior to use.
  4. Calculate the amount of ReadyFect TM that you will require. Use 3 μL of Axol
  5. ReadyFect TM per μg of DNA, as this is the optimal ratio (3:1) for Axol hNPCs.
  6. 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.
  2. Prepare your Axol ReadyFect TM solution by 0.375 μL to 30 μL of Axol ReadyFect TM in PBS according to guidelines in Table 1.
  3. Add DNA solution to the Axol ReadyFect TM 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 Complete Axol Neural Maintenance 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% CO 2 .
  7. Evaluate your transgene expression 24 to 72 hrs post-transfection.
  8. Schematic Overview of The Standard Transfection Protocol

Optimisation Steps

Although high transfection efficiencies can be achieved with the standard protocol, some optimization might be necessary in order to obtain optimal transfection efficiency.

Several parameters can be optimized including ratio of Axol ReadyFect TM to DNA, quantity of DNA used, cell density and incubation time.

Top Tip: Modify one parameter at a time while keeping the other parameters (cell number, incubation time etc.) constant. The two most critical variables are the ratio of Axol ReadyFectTM to DNA and the quantity of DNA.

1. Axol ReadyFect TM / DNA ratio: This is the most important parameter for optimization. First, maintain a fixed quantity of DNA (according to the size of your culture dish or cell number) and then vary the amount of Axol ReadyFect TM reagent as indicated in Table 2. You can test ratios from 1 to 6 μL of Axol ReadyFect TM reagent per 1 μg DNA.

2. Quantity of DNA: After optimization of the Axol ReadyFect TM / DNA ratio, proceed to adjust the amount of DNA required by maintaining a fixed ratio of Axol ReadyFect TM to DNA, and varying the DNA quantity over the suggested range (Table 3).

3. Axol ReadyFect TM : Several tests demonstrated that the use of PBS to prepare the DNA and Axol ReadyFect TM solutions leads to more reproducible transfections and in some cases higher efficiency, particularly with lower volumes of transfection reagent. PBS composition: 137mM NaCl, 2.7mM KCl, 1.5mM KH 2 PO 4 and 6.5mM Na 2 HPO 4 x 2 H 2 O; pH7.4.

4. Transfection Volume: To increase the efficiency of transfection you can reduce the transfection volume

Quality Control

To assure the performance of each Axol ReadyFect TM lot, we control the quality of each component using rigorous standard procedures. The  following in vitro assays are performed to guarantee the function, quality and activity of each component

Troubleshooting

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