1) Freshly dissociate organoids into single cells and then seed 500K in 500µl/well in a 48 well non-adherent plate with protamine sulfate with virus at MOI 3 for infection overnight.  (Patty and I have both stopped spin infecting the cells. This method seems to produce less cell death. I need to infect roughly 2x the number of cells that I will need for plating the next day to have enough for my experiments.)

2) The next day the cells are clumpy after infection so wash the cells in 5%CS DMEM and pass them through a 200µl pipette repeatedly until achieving a single cell suspension again.

3) Once acquired a single cell suspension, seed cells into either non-adherent plates for mammospheres or adherent plates for colony forming assays at a density of 20K/ml as described in Dontu et al. (When we plated dissociated cells from tumors, we plated at 5K/ml, thinking that more cells would form tumorspheres.)

The number of mammospheres that form following infection (or as freshly seeded cells) varies quite a bit among different patients.  Some patients form a ton of colonies, no suspension spheres, and medium numbers of mammospheres, etc.

4) Wait 7 days for mammosphere formation as described in Dontu et al.  (They don’t seem to grow larger (by eye) if you wait longer than that)

5) After 7 days, seed spheres  for the collagen gel experiments. Seed in the range of 1000 mammospheres/well in a 4-well chamber slide.  (To do this, multisize 3 wells of the 48 well plate, and plate 2-3 wells depending on the number that is calculate by multisizing).  Since replicates are similar for multisizing, this method seems to work well. 

The number of colonies that grow out of the mammospheres that are seeded are also patient sample dependent. The range is around 30-50% of mammospheres form some kind of colony. It seems that the patients that form the highest number of mammospheres have the lowest % of colony growth after seeding. By looking at the wells after seeding after each day you can see specifically which mammospheres are going to grow or not.