Im culturing MCF-7 (ATTC,USA) in EMEM (Mediatech, USA) supplimented with 5% FBS (PAA,USA) & 1% pen-strep (PAA,USA) incubated in 37degC, 5% CO2. After 24h, they turn pinkish and cloudy. The cells start to lyse, debrise formed and if I leave it for 48hr, all cell lysed, media get more alkaline. When I used 10% FBS (Im supposed to use this percentage in my research), media gets cloudy, 50% cells floated. If I try 15%, cells died in 24h. I checked old media's pH daily. pH climbed from 7.4 to 7.8 to 8.2 the following days.
Im using T75 flask, had to loosened the caps. If not cells near the cap will confluent but cells at the back of the flasks lysed. Suspected that the cells are not getting enough CO2.
I only get confluent cells, hassles-free if I gave the cells 5% FBS, seed at higher seeding density, loosen the cap, dont spray ethanol before keeping the flasks in the incubator & change media daily. No floating cells, no debrise, 80% confluence at 2days. But why? I cant keep doing this, I'll be using a lot of media quickly. Finding the balance is time consuming, I need to keep moving. I have similar problem with MDA-MB-231.
I really need help, guys.
Edited by bokashi farmer, 04 September 2010 - 09:21 PM.