9- On Top cells culture



Before splitting the cells, prepare the


Matrigel preparation


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)


Split the cells (see protocol “splitting and passing…”


Plating cells


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”