Transfection of Axol human Neural Progenitor Cells using Axol ReadyFect

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

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 ReadyFect^TM solutions should be at room temperature and be gently vortexed prior to use.

4.   Calculate the amount of Axol ReadyFect^TM that you will require. Use 3 μL of Axol ReadyFect^TM 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.

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 ReadyFectTM in 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.

Optimization 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 ReadyFectTM 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 ReadyFectTM / 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 ReadyFectTMreagent as indicated in Table 2. You can test ratios from 1 to 6 µL of Axol ReadyFectTM reagent per 1 µg DNA.

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

3.   Axol ReadyFectTM: Several tests demonstrated that the use of PBS to prepare the DNA and  Axol ReadyFectTM 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 KH2PO4 and 6.5mM Na2HPO4 x 2 H2O; pH7.4.

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

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.