TITLE: Cell culture, transfection and imaging
AUTHORS: Marianna Leonzino, Andrés Guillén-Samander, Pietro De Camilli

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>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 </span><a href="https://doi.org/10.1083/jcb.202010004" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1083/jcb.202010004</span></a><span style = ":;">.</span></div><br/></div>

[STEPS]
?. [General preparation]
Culture the COS-7 or HeLa (ATCC) cells at  and 5% CO2 in DMEM containing 10% FBS,  sodium pyruvate,  penicillin,  streptomycin and  L-glutamine (all from Gibco).
37 °C
100
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.
?. [General preparation]
For imaging experiments, seed the cells on glass-bottomed dishes (MatTek) at a concentration of 75x103 cells per dish and transfect transiently after  using FuGene HD (Promega).
?. [General preparation]
Image the cells for 36-48 hours after transfection.
?. [General preparation]
Just before imaging, remove the growth medium and replace with pre-warmed live-cell imaging solution (Life Technologies).
?. [General preparation]
Perform all live-cell imaging at  and 5% CO2.
37 °C
?. [General preparation]
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.
?. [General preparation]
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).
?. [Hypotonic shock experiments]
Replace the live-cell imaging solution with pre-warmed distilled water.
Suggestion: Minimal volume of live cell imaging media is placed in the dish to allow removal with a pipette in one single step. On the contrary, maximal volume of distilled water is added to allow the highest dilution of leftover ions in the dish.Warning: If media exchange is to be performed by hand, extreme care should be taken not to move the dish during the process. If substantial amount of live cell imaging media needs to be left in the dish to prevent movements during the aspiration procedure, a further media replacement step could be implemented to achieve maximal hypotonic shock.
?. [Experiments evaluating cytosolic Ca2+ changes]
Once a field of view with transfected cells is found, start the acquisition (generally use a rate of 0.5 Hz).
Cytosolic Ca2+ was monitored by the intensity of the RFP genetically encoded Ca2+ indicator for optical imaging (R-GECO; plasmid was a gift from R. Campbell, University of Alberta, Edmonton, AB, Canada; Addgene catalog no. 45494)
?. [Experiments evaluating cytosolic Ca2+ changes]
To acutely increase cytosolic Ca2+, add Thapsigargin (Life Technologies) to a final concentration of .
Note: Live-cell imaging solution (Life Technologies) contains   Ca2+.
?. [Experiments evaluating cytosolic Ca2+ changes]
Allow cells to recover for .
?. [Experiments evaluating cytosolic Ca2+ changes]
To decrease cytosolic Ca2+, add EGTA and BAPTA-AM (Thermo Fisher Scientific) to the medium to a final concentration of  and , respectively.
?. [Experiments evaluating cytosolic Ca2+ changes]
Image the cells for , then stop the acquisition.