X-tremeGENE™ HP DNA Transfection Reagent Protocol for transfection of SH-SY5Y cells

Protocol from: https://www.sigmaaldrich.com/GB/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/transfection-and-gene-editing/xtghp-general-protocol https://www.sigmaaldrich.com/GB/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/transfection-and-gene-editing/xtghp-general-protocol

Plate SH-SY5Y cells in 10mm²dish approximately 24 hours before transfection, making sure cells are at optimal concentration (70–90 % confluency).

Allow X-tremeGENE™ HP DNA Transfection Reagent, DNA and diluent (Opti:MEM^®I Reduced Serum Medium or serum-free medium) to warm to +15 °C to +25 °C, and vortex gently.1. Place diluent in a sterile tube.

1. Add plasmid DNA (2 µg) . Gently pipette up and down to mix.
2. Add 2 µL X-tremeGENE™ HP DNA Transfection Reagent to the diluted DNA.
3. Vortex the mixture.
4. Incubate for 15 minutes at +15 °C to +25 °C.
5. Add transfection complex to the cells in a dropwise manner.
6. Gently shake or swirl the wells or flasks to ensure even distribution over the entire plate.
7. Incubate cells for 72 hours before replacing with complete SH-SY5Y culture media (dx.doi.org/10.17504/protocols.io.bp2l617jzvqe/v1) supplemented with 400 µg/ml G418 antibiotics and incubated at 37°C and 5% CO₂ for selection.

Growth medium with selection reagent was replaced every 2-3 days. 1. After ensuring all mock transfections had died, colonies from the transfected cells were selected with sterile cloning cylinders.

1. Colonies were transferred to 6 well plates and maintain in growth medium containing G418.
2. Once confluent, cells were trypsinised and expanded into a 10mm²dish in 10 mL normal growth media.
3. Clones were frozen down or pelleted for characterisation.