You may have changed the medium to harshly.

Try to:

1) Coat your plate with ie 0.1% gelatin before seeding cells (add enough to cover bottom of well, leave for 15 min, remove excess liquid, allow to dry with lid off for 1h)

2) Be gentle when changing culture medium

- remove medium SLOWLY

- avoid cells drying out, work with only a few wells at the time

- add fresh medium SLOWLY and with low pressure.

- If using a 1mL pipet, gently press the pipet tip towards the side the well to spread the liquid. Personally, I prefer using a 10 mL serological pipet and a pipettor at the lowest speed setting.

Dear Karthik Bhat S ,

There are some morphological change occurs during the differentiation which turns the fibroblast cells into spherical shape and cell also divide after starting the differentiation so you might be seeing the changes.

This is a general Phenomenon.

IF cells are looking more Fibroblastic and peeled off from the well then it is due to stress condition.

Then there may be various reasons and suggestions:-

1. Passage no. of the cell line is very high So please use lower passage cells.

2. Maybe you started the treatment before full confluency, SO please start the differentiation after full confluency (You can incubate it for 72 hrs. also)

3. As earlier told by Jan-Inge Bjune , it may be due to changing the media harshly so be gentle and add the media slowly by using the wall of the well.

4. Change the plate or coat the well plate if you are still facing the problem because sometimes coating is also a problem.

Hope this will help to solve the problem.

Thank You