Jun 29, 2023
Generation of ATG3 KO Hela cells stably expressing HaloTag-LC3B
- 1Laboratory of James H. Hurley, University of California, Berkeley, CA
Protocol Citation: Xuefeng Ren 2023. Generation of ATG3 KO Hela cells stably expressing HaloTag-LC3B. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo3xq9v4o/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: June 11, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 83216
Keywords: ATG3 KO Hela cells, HaloTag-LC3B, ASAPCRN, generation of atg3 ko hela cell, atg3 ko hela cell, hela cell, lc3b this protocol details generation, lc3b, cell, expressing halotag, halotag
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000350
Abstract
This protocol details generation of ATG3 KO Hela cells stably expressing HaloTag-LC3B.
Attachments
736-1852.pdf
129KB
Materials
Buffers and reagents:
- Growth broth: LB broth
- Stbl3 competent E. coli (MacroLab, UC Berkeley)
- DMEM medium with GlutaMAX containing 10% FBS and 10% Pen-Strep.
pVSV-GaddgeneCatalog ##138479
pBS-CMV-gagpoladdgeneCatalog #35614
pMRX-IP-HaloTag7-LC3addgeneCatalog #184899
Qiagen Hi‐Speed MidiPrep kitQiagenCatalog #12643
DMEM, high glucose, GlutaMAX™ SupplementThermo FisherCatalog #10566016
Gibco™ Fetal Bovine Serum value heat inactivated (formerly USDA-approved in North America or qualiFisher ScientificCatalog #A5256801
Penicillin-Streptomycin (10,000 U/mL)Thermo Fisher ScientificCatalog #15140122
Opti-MEM™ I Reduced Serum MediumThermo FisherCatalog #31985070
TransIT®-LT1 Transfection ReagentMirus BioCatalog #MIR 2300
Lenti-X™ ConcentratorTakara Bio Inc.Catalog #631231
15-Dimethyl-15-diazaundecamethylene polymethobromide PolybreneMerck MilliporeSigma (Sigma-Aldrich)Catalog #H9268
Puromycin DihydrochlorideGold BiotechnologyCatalog #P-600
Procedures
15m
Transform each plasmid into Stbl3 competent cells for the propagation. Next day, pick up one colony to inoculate Overnight culture in LB medium with ampicillin.
Midi prep the cultures to purify plasmids (Qiagen).
Plate 5 x 106 HEK 293T cells on a 10 cm plate in DMEM medium.
HEK293T transfection:
Add retroviral packaging plasmids (pVSV-G, pBS-CMV-gagpol) and pMRX-IP-HaloTag7-LC3, 5 µg each in 1.5 mL warm Opti-MEM medium.
Add 45 µL of TransIT-LT1 transfection reagent (Mirus) and swirl.
Incubate at Room temperature for 00:15:00 .
15m
Add 1.5 mL dropwise into 10 cm HEK293T plate.
At 72 hours post-transfection, collect retroviral supernatant into a falcon tube.
Concentrate retroviral supernatant to 1 mL using Lenti-X concentrator (Takara) with the manufacturer instruction.
Retroviral transduction:
Plate 1 x 105 ATG3 KO HeLa cells into 12-well plate one day before.
Next day, titrate 100 µL , 200 µL , 400 µL of concentrated retroviral solution with 8 µg/mL Polybrene (Sigma) into target HeLa cells.
At 24 hours post-transduction, remove retroviral supernatant and replace with fresh DMEM complete medium with 2 µg/mL puromycin (GoldBio).
Perform Puromycin selection for two weeks. Confirm Halo-LC3B positive cells by FACS sorting.