TITLE: Protocol to culture mESCs (LSCB, UPLUT)
AUTHORS: Stefano Vianello, Mehmet Girgin, Giuliana Rossi, Matthias Lutolf

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Standard protocol used for culturing mouse embryonic stem cells (mESCs) in the Lutolf Lab, EPFL.</div></div>

[STEPS]
?. [Cell detachment]
Cell detachment:
?. [Cell detachment]
Using a vacuum pump+glass pipette,  aspirate all culture medium out of the well (but do not dry out the cells completely!)
?. [Cell detachment]
Add 2mL PBS-/- (against side of the well) ti wash medium traces, and then aspirate it out again
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Add 500uL Accutase, and let it digest ~ min,  , or until most cells are detached
0 Room temperature
You want to let Accutase digest until almost all cells have detached from the bottom of the well, even if they are still forming clumps in suspension. By the time you start collecting them up, they will have further dissociated into single cells.Softly tapping with your hand on the side of the plate will aid detachment of loosely-adherent cells.
While the digestion is taking place, and if not done previously, you can start preparing the solutions and tubes required later in the protocol.~6mL 10% Serum Medium to wash cells from the dish and, later, resuspend the pellet1 empty 15mL Falcon tube where you will collect the cells2mL "10%Serum 2i/LIF": 10% Serum Medium + 3uM CHIR99021 (1:1000 from our 3mM stock), 1uM PD0325901 (1:500 from our stock), 100u/mL LIF (1:1000 from our stock),
?. [Cell detachment]
With a P1000, collect all cells while breaking down clumps. To do so, pipette repeatedly up and down (without introducing bubbles) and use the ejected liquid to wash the  surface of the well. The goal is to collect as many cells as possible in suspension. At the end of the process, transfer to a 15mL Falcon tube.
?. [Cell detachment]
Further collect leftover cells by washing the well one last time with with 4.5mL 10%Serum Medium; then adding such suspension to the tube with the accutase/cell suspension. The total volume in the tube should now be ~5mL.
?. [Counting and plating]
Cell counting:
?. [Counting and plating]
Check that a nice pellet has formed at the bottom of the tube, and use a vacuum pump+glass pasteur pipette to remove all medium.
?. [Counting and plating]
Resuspend the pellet in 1mL 10%Serum Medium and pipette up and down for ~20 times. Change to a P200 and pipette up and down again for ~20 times to break down cells even finer.
?. [Counting and plating]
Using a P20 or a P10 pipette, pipette up and down the cell suspension solution for ~5 times, and load 10uL of the suspension into a chamber of the haemocytometer. Count cells and calculate their concentration in the original solution.
To count cells in the haemocytometer:tally the cells in each of the 4 big (4x4) squares at the four corners of the central crossdivide the resulting total by 4, to find the average number of cells per squaremultiply *10'000this is your # cells/mLif you then need e.g. 70'000 cells, divide 70k by the number you just found, to know how many uL of cell suspension to use
?. [Counting and plating]
Plate 70k cells in the new well of 2iLIF +10%Serum you had previously put to equilibrate in the incubator
?. [Counting and plating]
Slide the plate back and forth (cross pattern) across the surface of the laminar flow cabinet to distribute them evenly, and then leave the cells undisturbed in the incubator to allow them to attach
?. [Counting and plating]
Cells can be split every 2 or 3 days (choose one) without medium change in between. Daily checks are still recommended to monitor overcrowding and other possible issues.
?. [Cell detachment]
Centrifuge (1000rpm on our centrifuge),   ,
Centrifuge: 200 34
[min]
4 °C
While cells are centrifuging, add the 2mL of 10%Serum/2iLIF medium you prepared before to a new well of the 6well plate. Transfer this plate to the incubator to equilibrate it and have it ready to accept the new cells later on.