TITLE: Cell culture, transfection, immunocytochemistry, and imaging
AUTHORS: Michael G G Hanna, Pietro De Camilli

[DESCRIPTION]
This protocol is to help with the maintenance, transfection, immunocytochemistry, and imaging of adherent mammalian cells associated with the publication DOI above.

[STEPS]
SECTION: A. General cell culture
1. Culture COS-7 or HeLa (wild-type, parental control, and all knock-in lines) cells at 37 °C and 5 % in DMEM (Gibco, #11965118) containing 10 %, 1 millimolar (mM), 100 U/mL, 100 mg/mL (Gibco/LifeTech, #15140-122) and 2 millimolar (mM) (Gibco/LifeTech, #25030-081).

  
  
  
SECTION: A. General cell culture
2. Culture hTERT-RPE1 (wild-type, parental control, and all knockout lines) cells at 37 °Cand 5 % in DMEM/F12 (Gibco, #11330057) containing 10 %, 1 millimolar (mM), 100 U/mL, 100 mg/mL and 2 millimolar (mM).

  
 

SECTION: B. Cell transfection
3. For live-cell imaging experiments, seed cells on glass-bottom dishes (MatTek; 35mm) at the following concentrations: COS-7, 8.5x104; HeLa, 7.5x104; RPE1, 1.0x105.
SECTION: B. Cell transfection
4. Allow cells to adhere for 8-24 hours before being transiently transfected using FuGene HD (Promega) following manufacturer’s instructions.
 

SECTION: B. Cell transfection
5. Incubate transfection reagents with cells for 480 min-2160 min before imaging.

SECTION: C. Immunocytochemistry
6. For fixed-cell imaging experiments, seed cells on to glass coverslips (FisherBrand # 12542A) in a well of a 6-well plate (Corning #3516).

i. Perform seeding at the following concentrations for all fixed-cell experiments where cells are not additionally transfected: HeLa, 1.5x105; RPE1, 2.0x105.

ii. Perform seeding at the following concentrations for all fixed-cell experiments where cells are additionally transfected: HeLa, 7.5x104; RPE1, 1.25x105. Perform transfection as described above before fixation.
SECTION: C. Immunocytochemistry
7. Just before fixation, wash cells with 1 mL quickly.
SECTION: C. Immunocytochemistry
8. To each well, add 1 mL equilibrated at 37 °C. Perform fixation at 37 °C for a total of 10 min.
SECTION: C. Immunocytochemistry
9. Remove Fixation solution, and wash cells with TBS three 10-minute washes to ensure the stoppage and removal of fixation solution.
SECTION: C. Immunocytochemistry
9.1. Remove Fixation solution.

SECTION: C. Immunocytochemistry
9.2. Wash cells with TBS for 10 min.
SECTION: C. Immunocytochemistry
9.3. Wash cells with TBS for 10 min.
SECTION: C. Immunocytochemistry
10. After the last TBS wash, add 1 mL to each well for 10 min at 37 Room temperature.
SECTION: C. Immunocytochemistry
11. After solubilization is complete, add 1 mL for 60 min at 37 Room temperature.
SECTION: C. Immunocytochemistry
12. Dilute antibodies in AbDil buffer. 100 µL is used for each coverslip. Pipette the diluted antibody solution as a droplet onto parafilm in a humidity-controlled chamber.
SECTION: C. Immunocytochemistry
13. Invert coverslips onto the antibody droplet (cells facing down into solution) and place at 4 °C for 960 min -1440 min.

SECTION: C. Immunocytochemistry
14. Move coverslips into individual wells of a clean 6-well plate (cells now facing upwards) and wash three times with PBS wash buffer for 10 min each.
SECTION: C. Immunocytochemistry
9.4. Wash cells with TBS for 10 min.
SECTION: C. Immunocytochemistry
14.1. Move coverslips into individual wells of a clean 6-well plate (cells now facing upwards).
SECTION: C. Immunocytochemistry
14.2. Wash PBS wash buffer for 10 min.
SECTION: C. Immunocytochemistry
14.3. Wash PBS wash buffer for 10 min.
SECTION: C. Immunocytochemistry
14.4. Wash PBS wash buffer for 10 min.
SECTION: C. Immunocytochemistry
15. After the final wash, partially dry coverslips using an aspirator and invert (cells facing down) onto a drop of mounting medium (Invitrogen #P36935) on a glass slide (Electron Microscopy Sciences # 4951-001T). (Allow slides to cure as directed by the manufacturer of the mounting medium).

  

SECTION: C. Immunocytochemistry
16. After curing, permanently immobilize coverslips onto the glass slide using nail polish (Sally Hansen Advanced Hair and Nail).
SECTION: D. Imaging
17. Perform all imaging using an Andor Dragonfly system equipped with a plan apochromat objective (60x, 1.4 NA, oil) and a Zyla scientific CMOS camera.
SECTION: D. Imaging
18. Perform all live-cell imaging at 37 °C and 5 % in live cell imaging buffer (Invitrogen #A14291DJ).