Mar 19, 2024

Public workspaceCell culture, transfection, immunocytochemistry, and imaging

DOI
dx.doi.org/10.17504/protocols.io.5qpvokx3bl4o/v1
  • Nisha Mohd Rafiq1,
  • Pietro De Camilli2,3,4,5
  • 1University of Tuebingen;
  • 2Department of Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA.;
  • 3Department of Cell biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.;
  • 4Program in Cellular Neuroscience, Neurodegeneration and Repair.;
  • 5Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA.
Berrak Ugur
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DOI: dx.doi.org/10.17504/protocols.io.5qpvokx3bl4o/v1
Protocol Citation: Nisha Mohd Rafiq, Pietro De Camilli 2024. Cell culture, transfection, immunocytochemistry, and imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvokx3bl4o/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: March 12, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 96916
Keywords: ASAPCRN
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000580
Abstract
This protocol describes the maintenance, transfection, immunocytochemistry, and imaging of RPE1 and also transfection, immunocytochemistry, and imaging of iPSCs, i3 Neurons and DA neurons.
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Materials
Reagents:

  • ReagentLipofectamine™ 2000 Transfection ReagentThermo Fisher ScientificCatalog #11668019
  • ReagentLipofectamine™ Stem Transfection ReagentThermo Fisher ScientificCatalog #STEM00008
General cell culture for RPE1
General cell culture for RPE1
Grow hTERT-RPE1 cells in DMEM/F12 (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific), 1% glutaMAX, 1% penicillin-streptomycin. Keep cells at Temperature37 °C with 5% CO2 in an enclosed incubator.

Note
For general maintenance, when cells reach 80-90% confluency, detach them from the dish with Trypsin and dilute 1:10-20 in a new dish.

Cell transfection for RPE1
Cell transfection for RPE1
5d
5d
For live-cell imaging experiments, seed the cells on glass-bottom dishes (MatTek; 35mm) at the concentrations ranging from 1-2 × 105 cells.
For RPE1: allow the cells to adhere forDuration08:00:00 -Duration24:00:00 before being transiently transfected using Amount4 µL Lipofectamine™ 2000 Transfection Reagent (Invitrogen) in Opti-MEM media, mix them with the respective plasmids (Amount1 µg -Amount2 µg ) and visualize after Duration48:00:00 .
3d
For cilia generation, serum-starve the cells in DMEM/F12 media (without FBS) for Duration48:00:00 .

2d
Cell transfection for iPSCs, i3Neurons and DA Neurons
Cell transfection for iPSCs, i3Neurons and DA Neurons
2d
2d
For live-cell imaging experiments, seed cells on glass-bottom dishes (MatTek; 35mm) at the concentrations ranging from 3-5 × 105 cells.
UseAmount2.4 µL of Lipofectamine™ Stem Transfection Reagent (Invitrogen) in Opti-MEM media with respective plasmids (Amount1 µg -Amount3 µg ) and visualize at least Duration48:00:00 later.

2d
Immunocytochemistry of RPE1, iPSCs, i3Neurons and DA Neurons
Immunocytochemistry of RPE1, iPSCs, i3Neurons and DA Neurons
2h 7m
2h 7m
For fixed-cell imaging experiments, seed cells on glass-bottom dishes (MatTek; 35mm) at the concentrations ranging from 1-3 x 105 cells.

  • For immunofluorescence visualization, fix the cells with 4% (v/v) paraformaldehyde (Electron Microscopy Sciences) in 1× phosphate-buffered saline (PBS) for Duration00:20:00 .

20m
Wash cells thrice in PBS.
Wash
Perform cell extraction with 0.25-0.5% (v/v) Triton X-100 in PBS for Duration00:10:00 .

10m
Wash cells thrice in PBS.
For removal of free aldehyde groups, quench the cells with freshAmount1 µL sodium borohydride (Sigma-Aldrich) in PBS for Duration00:07:00 , and then wash thrice in PBS.

7m
Further block the cells for Duration00:30:00 in 5% bovine serum albumin (BSA, Sigma-Aldrich) in PBS and then incubate DurationOvernight at Temperature4 °C with the respective antibodies listed in Table S1.

30m
Incubation
Overnight
Wash the cells with PBS thrice the following day and incubate with Alexa Fluor-conjugated secondary antibodies (Thermo Fisher Scientific) for Duration01:00:00 at TemperatureRoom temperature .

1h
Wash the cells thrice in 1×PBS.
Use DAPI (Thermo Fisher Scientific) for nuclear staining, when necessary.
Imaging
Imaging
For live imaging, maintain cells in a caged incubator with humidified atmosphere (5% CO2) at Temperature37 °C .
Note
The Yokogawa spinning disk field scanning confocal system with microlensing (CSU-W1 SoRa, Nikon) controlled by NIS elements (Nikon) software was used for imaging. Excitation wavelengths between 405-640 nm, CFI SR Plan ApoIR 60XC WI objective lens and SoRa lens-switched light path at 1x, 2.8x or 4x were used. SoRa images were deconvolved using the Batch Deconvolution (Nikon) software.