Hi everyone, I have a question please. We have our BMDM ready, but it did not respond LPS (100ng/ml)and IL4(25 ng/ml) very well. I wonder if anyone have any suggestions or experience on the polarization and the assay that used to measure the polarization please.
We are using MCSF (50ng/ml)to differentiate mouse marrow cells. On Day6, we use CD11b and F4/80 staining to check the differentiation. We always get more than 98% percent of staining positive cells. We have isotope control, which shows negative staining results.
Then we seed BMDM into 96 well plate overnight, and change the media at the other day, and treat them with LPS and IL4 for 24 hours.
Then we use Griess assay and Urea assay to check for M1 and M2 activation respectively. The assays works twice, however, we can not repeat it anymore.
looking forward to any suggestions please!
Thanks a lot!
In my experience, BMDM can be washed off when you change the medium in the 96 well plate. Do you use a western blot tip to aspirate the medium with minimal disruption to the cells?
The number of cells present in the well of a 96 well plate may not be high enough to detect NO or urea in your assays. When carrying out a similar experiment, I used 6 well plates to ensure adequate numbers of M1 and M2 macrophages before carrying out Griess assay, ELISA, and qPCR.
Hi Martin,
Thank you very much for your suggestions~
I am using normal tips to aspirate the media through the edge of the well, it should be fine for the cells I think...
Yes, you are right, the number of the cells may be the reason... I am gonna try more cells~
Thank you very much! :)
Best
Wang
- Badges
- Science topic
- Similar topics
- Medicine
- Disease
- Disease Models
- Animal Models
- Rodent Models
- Mouse Models