HT22 cells are difficult to transfect, so selecting the proper transfection reagent is critical. Here are some good options :

Choosing the Right Transfection Reagent

HT22 cells are difficult to transfect, so selecting the proper transfection reagent is critical. Here are some good options:

-Lipofectamine 3000 (Thermo Fisher): This is widely used and has been shown to provide high transfection efficiency in many cell types, including neurons.

-Lipofectamine 2000 (Thermo Fisher): A more cost-effective option but still effective for HT22 cells.

-PEI (Polyethylenimine): Another alternative that is cost-effective and works well with neuronal cells.

Pro tip: Lipofectamine 3000 is generally preferred for HT22 cells due to its efficiency, especially when dealing with plasmids or RNA.

Reagent Preparation

-DNA or RNA Plasmids: Ensure the plasmids are of high quality. You can use a standard plasmid extraction kit (like the Qiagen Midi Kit). Endotoxin-free plasmid preparations are ideal for transfection to minimize toxicity.

-Opti-MEM: For dilution of the transfection reagents and DNA. Always use Opti-MEM or another serum-free medium for the transfection mixture (not regular culture medium, as serum can interfere with transfection efficiency).

-Transfection Reagent: Prepare the reagent exactly as per the manufacturer’s instructions.

Cell Preparation :

Cell Density: Seed HT22 cells the day before transfection, at about 60-70% confluency. Too low confluency can reduce efficiency, and too high confluency can lead to toxicity.

The day before transfection, make sure that cells are healthy and growing actively (not in a stationary phase).

Transfection Procedure

Step 1 : Dilute your plasmid (typically 1-2 µg per 60 mm dish) in Opti-MEM medium (no serum).

Step 2 : Dilute the transfection reagent in Opti-MEM according to the manufacturer's recommendations (usually around 3-5 µl reagent per µg of DNA).

Step 3 : Mix the plasmid and transfection reagent separately and then combine them gently. Let the mixture incubate for about 15-20 minutes at room temperature to allow complexes to form.

Step 4 : After incubation, add the transfection mix to your cells. Gently swirl the plate to distribute the transfection mixture evenly.

Post-Transfection Care

-Incubation: After transfection, return the cells to the incubator (typically at 37°C and 5% CO2).

-Serum: You can add serum back to the medium 6-12 hours after transfection. However, if your reagent suggests serum-free medium for 24 hours, follow that guideline for best results.

-Antibiotics: Avoid using antibiotics in the culture medium immediately post-transfection, as they can interfere with cell survival and transfection efficiency.

Optimizing Transfection Efficiency

Optimize DNA Amount: Too much DNA can increase toxicity, while too little may lower efficiency. Start with around 1-2 µg of DNA per well in a 6-well plate, and adjust according to the outcome.

Use of a Plasmid with a Reporter Gene: If you're not already using one, consider incorporating a reporter gene like GFP or Luciferase to monitor transfection efficiency.

Timing: The transfection efficiency of HT22 cells can vary depending on the time of transfection. 24-48 hours after transfection is often the optimal time for analyzing expression, but it can vary depending on the plasmid.

Harvesting Post-Transfection

If you’re expressing a reporter gene like GFP, use a fluorescent microscope to monitor expression.

For protein or RNA expression analysis, perform your downstream applications (e.g., western blotting, qPCR, or immunocytochemistry) 24-48 hours post-transfection, depending on your plasmid's expression rate.

Dear Nada Ahmed Selim,

I totally agree with Nicolas’ post; the tips are really good!

However, I would like to jump-in to propose a real alternative to classic transfection that could be a game changer for HT-22 genetic modification. And this would be the Magnetofection*** technology. Among the reagents we have developed based on this technic (and also, of course, on classic chemicals) there is NeuroMag that was recently demonstrated to be efficient for HT22 transfection with DNA.

You can refer to the publication by Paes D. et al, Cell Mol Life Sci. 2023 Jun 12;80(7):178. doi: 10.1007/s00018-023-04804-w.

It is true that NeuroMag was originally developed to specifically transfect primary hippocampal and cortical neurons but since almost two decades now, it has been successfully demonstrated to be highly efficient on almost every type of primary neurons (cortical, hippocampal, motor neurons…) and also “neuronal cell types”, such as astrocytes, oligodendrocytes, glial cells as well as various other cell types such as HT22.

This is why I think it could be worth using it.

you can have more infos at: https://ozbiosciences.com/neurons/52-50-neuromag-neuron-transfection-reagent.html#/22-capacity-200_l

Would you like to give NeuroMag a try, please do not hesitate to contact me via ResearchGateor directly at: [email protected]; I will be glad to send you a sample !

best regards,

Cedric

*** The Magnetofection technology uses a magnetic field to attract and concentrate complexes of magnetic nanoparticles and nucleic acid onto the cell surface as demonstrated by Grześkowiak BF et al, Pharm Res. 2014 Jul 18. (http://www.ncbi.nlm.nih.gov/pubmed/25033763).