I am polarizing macrophage from THP-1 cells, then treat macrophage with conditional medium. But the gene expression is so unstable that I can’t properly examine the problems.
The experiment method and three sets of the result is show down below (experiment is done by qPCR).
I had properly managed the concentration of stimuli, make sure they’re properly mixed when treatment, I also make sure the condition of THP-1 is normal when seeding.
Also, I found oud THP-1 have a tendency to grow faster through time, although seniors in lab don’t think it’s a problem.
Hope someone could provide me some advice of experiment and how to improve when executing the experiment. Thanks!
1. Count cell number before plating cell, each condition needs triplicates and stimulate cells at the same time period ( plating at 10:00 am and stimulate after 24h, keep this 24h time gap for each experiment) to make sure each time they are the same amount.
2. LPS concentration is too high for THP-1, it will make big variants even though a tiny volume difference. Premix stimulus with medium first and then add equal amount to each well.
3. Make sure to use the same batch of conditional medium. Because components in conditional medium may vary a lot from due to the cell numbers, harvest time and even cell status.
4. Try to add more markers, M1(CD80, CD38), M2 (CD163)
Good luck!
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