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.
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”