Apr 18, 2022
Cell culture, transfection, and imaging
- Will Hancock-Cerutti1,2,3,
- Pietro De Camilli1,3
- 1Departments of Neuroscience and of Cell Biology, Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 2Interdisciplinary Neuroscience Program and MD-PhD Program, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 3Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
Protocol Citation: Will Hancock-Cerutti, Pietro De Camilli 2022. Cell culture, transfection, and imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq41j3vk5/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: July 08, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 51390
Keywords: Cell culture, Transfection, Spinning-disk confocal, Fluorescent microscopy, ASAPCRN
Abstract
This protocol describes general procedures for culturing HeLa cells, transient transfection, and imaging using an Andor Dragonfly spinning disk confocal system.
Attachments
dxwubkjz7.pdf
142KB
Materials
DMEM Solution:
| A | B | |
| FBS | 10% | |
| Penicillin | 100 U/ml | |
| Streptomycin | 100 mg/ml | |
| L-glutamine | 2 mM |
* (all from Gibco)
General preparation
General preparation
Culture the HeLa-M cells at 37 °C in 5% CO2 and DMEM containing 10% FBS, 100 U/ml penicillin, 100 mg/mL streptomycin, and 2 millimolar (mM) L-glutamine (all from Gibco).
Note
Note: For general maintenance, when cells reached 80-90% confluency, they were deattached from the dish with Trypsin and diluted 1:20 in a new dish.
For live-cell imaging experiments, seed the cells on glass-bottomed dishes (MatTek) at a concentration of 35,000 cells per dish and transfect after 24:00:00 using FuGene HD (Promega) in Opti-MEM (Gibco).
1d
Image the cells 24:00:00 after transfection.
1d
Just before imaging, remove the growth medium and replace with prewarmed live-cell imaging solution (Life Technologies).
For lysotracker experiments, incubate the cells in 50 nanomolar (nM) LysoTracker Red DND-99 (ThermoFisher) in complete DMEM for 00:30:00 , wash twice with media, then image in live-cell imaging solution.
30m
Perform all live-cell imaging at 37 °C and 5% CO2.
Perform spinning-disk confocal microscopy using an Andor Dragonfly system equipped with a plan apochromat objective (63×, 1.4 NA, oil) and a Zyla scientific CMOS camera.
For any given experiment, use the same exposure time, laser power, and gain for image acquisition to allow for quantitative comparison.
Imaging of cells stably expressing STING-GFP
Imaging of cells stably expressing STING-GFP
3d 14h
3d 14h
Generate the cells stably expressing STING-GFP as described elsewhere.
Culture the stable STING-GFP HeLa-M cells at 37 °C in 5% CO2 and DMEM containing 10% FBS, 100 U/ml penicillin, 100 mg/mL streptomycin, and 2 millimolar (mM) Lglutamine (all from Gibco).
For experiments using siRNA, transfect 60 pmols of the indicated siRNA using 6 µL Lipofectamine RNAiMax (ThermoFisher) in Opti-MEM (Gibco) per dish according to manufacturer protocol. Image the cells 72:00:00 after siRNA transfection.
3d
For experiments using cGAMP, transfect 50 μg/L of cGAMP using 18 µL Lipofectamine RNAiMax (ThermoFisher) in Opti-MEM (Gibco) per dish according to manufacturer protocol. Image the cells 14:00:00 after transfection.
14h