It is normal to see the cell dead. To increase the protein level and extend the incubation period, transfer cells into 32degree incubator can help.

(see manual from thermo fisher)

Thank you for your reply Lo Tzu Yu

But, I already followed the instruction from Thermo.

I transfected the DNA to the cell and incubated it at 37℃ for 18-22 h (Day0) as manual.

I added the feed and enhancer to the cell (Day1).

And then, I transfered it 32℃ incubator. But the cells were dead at day3.

What is the viability when you collect your conditioned media? I think it should be okay to be around 60 to 80%

Thermo provides positive in its kit. maybe you can compare the expression level with it?

Lo Tzu Yu Plz check attached viability image. I got the cell stock from another labs, so we donot have control plasmid, I'll order the control vector.

Thank you :)

Dear Soi Yun

did you growth the cells in an orbital shaker with 120rpm agitation?

Is quite normal observe a viability reduction at the end of the culture also if your cells seems to start do dye little earler.

In generall as reported in the manual (maxtiter protocoll) for 8 day week culturem I add the feed 2 times at the day 1 (with the enancher) and at day4. In generally i'm trasfercting 25ml in a 125ml shake flask and i add 4ml of feed each time.

good luck

Manuele

Dear Manuele Martinelli and Lo Tzu Yu

Thank you for your reply :D

I found big problem on the CO2 incubator.

I re-tried again with your suggestions and in another incubuator, i could express the protein well. Thank you so much again!