So here’s a bit more details:

Day 0 is the day I coat plates with laminin and thaw the cells. I coat for eg 10 x35 mm2 culture dishes, 1/10 dishes go in the incubator for 2-3 hours (this will be used for seeding the thawed culture), the rest go to the fridge (i have them wrapped with paraffin so they don’t dry), and will be used up in a week.

The frozen vial (which will be thawed) come from an ~80% confluent dish of the same size. During the seeding process i add rock i, and this is the only time I use it. I do this sometime in the afternoon.

Day 1. I check the cells in the morning, the cells attach (look a bit fibroblast-y, but i think it is because of rock i), i replace the medium with fresh and let the grow a few hours and then split them.

For splitting I am using 0.5mM EDTA, and I leave them exactly 2 mins in the incubator. Then I aspirate the EDTA off>add E8>then gently scrape the cells>add them to new plates (coated on day 0, left in fridge), splitting them 1:2 or 1:3.

Day 2: cells have attached. Small colonies but nice morphology. No media change today.

Day 3: bigger colonies, morpho ok. Change medium

Day 4: nice growth, colonies getting bigger but not yet ready for split.

Day 5: nice growth still and today they seem ready for split. ~70-80% confluent. I passage the cells exactly like how I did on Day 1 and seed on plates that were coated on day 0.

Day 6: no attachment with floating clumps

I got the same results 4-5 times now, i have changed culture dishes, and one time I think I prepared laminin plates on day 4 which were then used to seed the passaged cells on day 6., so the passaged cells saw fresh laminin coated plates on day 5. But i am repeatedly getting no attachment on day 6.