Thaw on the ice O/N (keep it in the fridge)
1 bottle thawed: 1 month at 0C or refrozen once
(make aliquots)
Using cold tips and cold dishes can help to spread
more homogeneously the matrigel
Put the right volume of matrigel (0.5ml fro a 35mm
dish) in the center of the dish without bubble, and then spread with a tip
(blue or yellow depending on the surface)
20 min. in the incubator (37 degree)
1-
Take
only number of cells you need for each kind of plates
2-
Centrifuge
5min at 800 rpm
3-
Aspirate
medium
4-
Resuspend
cells with the half volume of H14 medium you will need according to the size
of the dishes (0,5 ml for 35 mm )
5-
Put
the cells on top of the pre-coated dish.
For the on top culture, there is to way to process
(A: Sophie and Eva, B: Hong)
6A- Wait 2 min.
7A- Adjust the final volume drop by drop with
media+add+/-matrigel 10% (you will have matrigel 5% final), for example for 35
mm dish: mix 0.45 ml media+add and 0.05 matrigel, and add it to the cells.
Or
6B- Wait 15 min to 30 min for the cells to be
settled,
7B- Prepare media+additives+5 % matrigel
8B- Remove all the media (with cells that are still
floating)
8B- Add drop by drop, the right volume of
media+additives+5 % matrigel to each “on top dish”