May 23, 2022

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

DOI
dx.doi.org/10.17504/protocols.io.rm7vzy54rlx1/v1
  • 1Department of Clinical and Movement Neurosciences, Queen Square Institute of Neurology, University College London (UCL)
Laura Smith
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzy54rlx1/v1
Protocol Citation: Laura Smith 2022. X-tremeGENE™ HP DNA Transfection Reagent Protocol for transfection of SH-SY5Y cells. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzy54rlx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: April 28, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 61602
Keywords: ASAPCRN
Abstract
SH-SY5Y cells were transfected with a pcDNA3.1 vector plasmid. Stable transfection was performed using the XtremeGENE reagent for 72 hours, and 400 µg/ml G418 antibiotics used as the selection marker . Colonies were selected and expanded for routine culture in growth media supplemented with G418.
Safety warnings
  • This is a genetic manipulation procedure and as such must have been approved by the GM Committee to cover the plasmid, inserted DNA, cells to be transfected and person performing the experiment.
  • Only specific rooms are covered for these experiments (see the departmental safety codes of practice).
  • Make sure you are familiar with these safety documents, and in particular the protocols relating to spillages and decontamination.
Cell Preparation for Transfection
Cell Preparation for Transfection
Plate SH-SY5Y cells in 10mm2dish approximately 24 hours before transfection, making sure cells are at optimal concentration (70–90 % confluency).
Transfection
Transfection
  1. 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.
  2. Place diluent in a sterile tube.
  3. Add plasmid DNA (2 µg) . Gently pipette up and down to mix.
  4. Add 2 µL X-tremeGENE™ HP DNA Transfection Reagent to the diluted DNA.
  5. Vortex the mixture.
  6. Incubate for 15 minutes at +15 °C to +25 °C.
  7. Add transfection complex to the cells in a dropwise manner.
  8. Gently shake or swirl the wells or flasks to ensure even distribution over the entire plate.
  9. 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.
Culturing transfected SH-SY5Y cells
Culturing transfected SH-SY5Y cells
  1. Growth medium with selection reagent was replaced every 2-3 days.
  2. After ensuring all mock transfections had died, colonies from the transfected cells were selected with sterile cloning cylinders.
  3. Colonies were transferred to 6 well plates and maintain in growth medium containing G418.
  4. Once confluent, cells were trypsinised and expanded into a 10mm2dish in 10 mL normal growth media.
  5. Clones were frozen down or pelleted for characterisation.