Cell culture, transfection and imaging
Marianna Leonzino, Andrés Guillén-Samander, Pietro De Camilli
Abstract
This protocol details the general preparation of cells for imaging and also for imaging experiments involving cellular hypotonic shock and cytosolic Ca2+ changes as they were performed in http://doi.org/10.1083/jcb.202010004.
Attachments
dn3vbgtzx.pdf
Steps
General preparation
Culture the COS-7 or HeLa (ATCC) cells at 37°C and 5% CO2 in DMEM containing 10% FBS, 1millimolar (mM) sodium pyruvate, 100U/ml penicillin, 100mg/mL streptomycin and 2millimolar (mM) L-glutamine (all from Gibco).
For imaging experiments, seed the cells on glass-bottomed dishes (MatTek) at a concentration of 75x103 cells per dish and transfect transiently after 6h 0m 0s using FuGene HD (Promega).
Image the cells for 36-48 hours after transfection.
Just before imaging, remove the growth medium and replace with pre-warmed live-cell imaging solution (Life Technologies).
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.
Identify the cells to be imaged by scanning the dish.
Hypotonic shock experiments
Once a field of view with transfected cells is found, start the acquisition (generally use a rate of 12 frames/minutes).
Replace the live-cell imaging solution with pre-warmed distilled water.
Experiments evaluating cytosolic Ca2+ changes
To acutely increase cytosolic Ca2+, add Thapsigargin (Life Technologies) to a final concentration of 2micromolar (µM).
Allow cells to recover for 0h 10m 0s.
To decrease cytosolic Ca2+, add EGTA and BAPTA-AM (Thermo Fisher Scientific) to the medium to a final concentration of 4millimolar (mM) and 10micromolar (µM), respectively.
Image the cells for 0h 10m 0s, then stop the acquisition.
