May 23, 2024
Version 1
Transfection of HT22 cells with Lipofectamine LTX V.1
This protocol is a draft, published without a DOI.
- Signe Goeke1
- 1Max Planck Institute
Protocol Citation: Signe Goeke 2024. Transfection of HT22 cells with Lipofectamine LTX. protocols.io https://protocols.io/view/transfection-of-ht22-cells-with-lipofectamine-ltx-dd8429yw
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: May 23, 2024
Last Modified: May 23, 2024
Protocol Integer ID: 100348
Abstract
Transfection of HT22 cells
Day 0
Day 0
Plate HT22 cells in 6 well plate: 250K per well
If you want to perform transfection next day plate 500K.
Day 1
Day 1
Check cells --> less than ~60-70% confluent wait another day
Day 3
Day 3
Dilute the optimized amount of plasmid DNA in 500 μl Opti-MEMI Reduced Serum Medium (no A/A, no FCS). Mix thoroughly.
Mix PLUS™ Reagent gently before use, add 2.5uL volume of PLUS™ Reagent directly to the diluted DNA. Mix gently and incubate for 5 minutes at room temperature.
Mix Lipofectamine LTX gently before use, and add 5uL directly to the diluted DNA. Mix thoroughly.
Incubate for 30 minutes at room temperature (DNA-lipid complexes are stable for 6 hours at room temperature)
Meanwhile: Add 2mL of antibiotic-free medium containing 5% FCS
Add ~500 μl DNA-lipid complex (step 4 above) dropwise to the well containing cells. Mix gently by rocking the plate back and forth.
Incubate the cells at 37°C in a CO2 incubator for 18–48 hours prior to testing for transgene expression.
Change medium after 4–6 hours to regular medium (with A/A and 10% FCS)