TITLE: Cell culture, transfection, immunocytochemistry, and imaging
AUTHORS: Nisha Mohd Rafiq, Pietro De Camilli

[DESCRIPTION]
This protocol describes the maintenance, transfection, immunocytochemistry, and imaging of RPE1 and also transfection, immunocytochemistry, and imaging of iPSCs, i3 Neurons and DA neurons.

[STEPS]
SECTION: General cell culture for RPE1
1. 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 37 °C with 5% CO2 in an enclosed incubator. 

 

SECTION: Cell transfection for RPE1
2. For live-cell imaging experiments, seed the cells on glass-bottom dishes (MatTek; 35mm) at the concentrations ranging from 1-2 × 105 cells.
SECTION: Cell transfection for RPE1
3. For RPE1: allow the cells to adhere for480 min-1440 minbefore being transiently transfected using 4 µL Lipofectamine™ 2000 Transfection Reagent (Invitrogen) in Opti-MEM media, mix them with the respective plasmids (1 µg -2 µg) and visualize after 2880 min.
SECTION: Cell transfection for RPE1
4. For cilia generation, serum-starve the cells in DMEM/F12 media (without FBS) for 2880 min. 

SECTION: Cell transfection for iPSCs, i3Neurons and DA Neurons
5. For live-cell imaging experiments, seed cells on glass-bottom dishes (MatTek; 35mm) at the concentrations ranging from 3-5 × 105 cells.
SECTION: Cell transfection for iPSCs, i3Neurons and DA Neurons
6. Use2.4 µL of Lipofectamine™ Stem Transfection Reagent (Invitrogen) in Opti-MEM media with respective plasmids (1 µg-3 µg) and visualize at least 2880 min later.

SECTION: Immunocytochemistry of RPE1, iPSCs, i3Neurons and DA Neurons
7. 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 20 min.

SECTION: Immunocytochemistry of RPE1, iPSCs, i3Neurons and DA Neurons
8. Wash cells thrice in PBS. 
SECTION: Immunocytochemistry of RPE1, iPSCs, i3Neurons and DA Neurons
9. Perform cell extraction with 0.25-0.5% (v/v) Triton X-100 in PBS for 10 min. 

SECTION: Immunocytochemistry of RPE1, iPSCs, i3Neurons and DA Neurons
10. Wash cells thrice in PBS. 
SECTION: Immunocytochemistry of RPE1, iPSCs, i3Neurons and DA Neurons
11. For removal of free aldehyde groups, quench the cells with fresh1 1524sodium borohydride (Sigma-Aldrich) in PBS for 7 min, and then wash thrice in PBS. 

SECTION: Immunocytochemistry of RPE1, iPSCs, i3Neurons and DA Neurons
12. Further block the cells for 30 min in 5% bovine serum albumin (BSA, Sigma-Aldrich) in PBS and then incubate 10 min at 4 °C with the respective antibodies listed in Table S1. 

SECTION: Immunocytochemistry of RPE1, iPSCs, i3Neurons and DA Neurons
13. Wash the cells with PBS thrice the following day and incubate with Alexa Fluor-conjugated secondary antibodies (Thermo Fisher Scientific) for 60 min at Room temperature.

SECTION: Immunocytochemistry of RPE1, iPSCs, i3Neurons and DA Neurons
14. Wash the cells thrice in 1×PBS.
SECTION: Immunocytochemistry of RPE1, iPSCs, i3Neurons and DA Neurons
15. Use DAPI (Thermo Fisher Scientific) for nuclear staining, when necessary.
SECTION: Imaging
16. For live imaging, maintain cells in a caged incubator with humidified atmosphere (5% CO2) at 37 °C.