Dec 03, 2024
Stable Cell Line Generation – RAW 264.7
- Amanda Bentley-DeSousa1,2,3,4,5,
- Shawn Ferguson1,2,3,4,5,6
- 1Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 2Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 3Program in Cellular Neuroscience, Neurodegeneration and Repair;
- 4Wu Tsai Institute Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 5Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA;
- 6Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA
Protocol Citation: Amanda Bentley-DeSousa, Shawn Ferguson 2024. Stable Cell Line Generation – RAW 264.7. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm9nr5l3p/v1
Manuscript citation:
https://doi.org/10.1101/2023.10.31.564602
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: December 02, 2024
Last Modified: December 03, 2024
Protocol Integer ID: 113412
Keywords: RAW 264.7, piggybac, stable expression
Funders Acknowledgements:
Aligning Science Across Parkinson’s Disease
Grant ID: ASAP-000580
Abstract
This protocol details steps taken to create stably expressing cell lines in RAW 264.7 cells.
Materials
DMEM (Thermo Fisher Scientific, 11965-092)
FBS (Thermo Fisher Scientific, 16140-071)
Penicillin/Streptomycin (Thermo Fisher Scientific, 15140122)
CellStripper (Corning, 356230)
Lipofectamine 2000 (Invitrogen, 11668019)
FuGene HD (Promega, E2311)
Opti-Mem (Thermo Fisher Scientific, 31985062)
Puromycin (Gibco A11138-03)
Blasticidin (Invivogen ant-bl)
Day 0
Day 0
Plate 2.5 x 10^5 RAW 264.7 cells per well in a 6-well dish.
Day 1
Day 1
30m
30m
Transfect cells using Lipofectamine 2000 or FuGene HD. Ensure to use a 1:2 ratio of Piggybac transposon plasmid:gene-of-interest plasmid for a total of 1 uG DNA.
For Lipofectamine 2000 (HALO-LRRK2):
In Tube 1 combine 150 µL Opti-MEM and 8 µL Lipofectamine 2000 then incubate for 00:05:00 . In Tube 2 combine 150 µL Opti-MEM and 1 uG total DNA (see above) then incubate for 00:05:00 . Combine tube 1 and tube 2. Incubate for 00:20:00 . Add drop-wise to 1 well containing 1.7 mL of media.
30m
Day 3
Day 3
Change the media.
Day 4
Day 4
Add media containing selection-specific antibiotics.
Use Puromycin (3.5 uG/mL) or Blasticidin (10 uG/mL).
Day 6-7
Day 6-7
Change the media.
Day 8+
Day 8+
After cells have recovered from antibiotic selection, plate single cells into 96-well dishes to obtain clonal cell lines. After subculturing, confirm cell lines via immunoblotting.