Hello,

I was used to freeze Min6 in 10% DMSO and 90% DMEM High Glucose (The DMEM was previously supplemented with 15% FBS).

In order to revive them, I plated cells in 25ml Flasks and kept them in culture (changing medium and splitting them) for 1-2 weeks.

I never stored them at so high passage because in my hands after the 40s passage they were more sensitive to stresses. In fact I found on many papers that Min6 were usued from passage 15/19 to passage 40/45.

Thanks sir,

Can you provide me detail protocol of freezing and thawing cell ?

As i got min6 on p59 from nccs and i stored them after spliting on p61, 62.

But the problem i am facing again and again that my cells does not get attach on surface.

For freezing i use 10% DMSO, 20% FBS, 70%DMEM (freezing media composition) and during reviving my cell first i thaw on 37C then in 5ml growth media centrifuge on 800rpm for 3 min then add cell in 5ml growth media in T25flask.

My growth media composition is DMEM 25mM 15%fbs 1%penstrip.