Hi,
I was experimenting with different amount of seeded RAW 264.7 cells incubated with LPS to find out which seeding density to use for my upcoming experiments.
I was using the 10k, 20k, 40k, 80k and 100k cells in 96 wells.
During the experiment, I noticed that post LPS incubation, the wells containing 80k and 100k cells that were treated with LPS turned yellowish indicating a decrease in pH. The 80k and 100k wells without LPS remained the same color as the well with only culture media and no cells.
Does anybody know what is causing the drop in pH?
This was my experiment.
1) Seed 200ul of corresponding amount of cells (i.e. 10k, 20k, 40k, 80k, 100k) in 96 well. Incubate 24 hours in 37C, 5% CO2 to stabilize.
2) Aspirate media and cell debris. Add fresh 200ul of media.
3) Add 22ul of LPS 10ug/ml to reach final concentration of 1ug/ml LPS. Incubate for 24 hours in 37C, 5% CO2.
Everything was done with technical triplicate.