I realize this is a question that has probably been previously addressed!
We are currently trying to differentiate monocytes to to macrophages using autologous serum from the same donor. The general protocol we follow is as such:
1) standard PBMC isolation using Ficoll
2) Plating isolated PBMC fraction in a non TC plate for 30 min-1 hour to allow monocyte adhesion
3) Washing off adherent cells with DPBS
4) adding RPMI+ pen strep + 10-40% autologous human serum and culturing withour disturbance for 6 days.
However, within the first few days itself the cells begin to look pretty terrible and almost none survive and/or differentiate.
I was wondering what I could start with in terms of troubleshooting prior to resorting to magnetic isolation of monocytes or using any sort of growth factor within the medium.
I'd be very thankful for any suggestions. Finally, just as a side, although the established literature suggests autologous serum, is there much of an effect to be expected if say one were to use autologous plasma? I ask as we already have some reserves of plasma that could be used for additional attempts.
https://www.researchgate.net/post/Differentiation_of_primary_human_monocytes_to_monocyte_derived_macrophages
Monocytes will die within 2-days if they don't get any stimulation. To differentiate macrophages, you need to stimulate monocytes with M-CSF or GM-CSF.
Can anybody recommend specific seeding densities? What would be ideal for optimum differentiation? in both 24 and 96 well-playteformats
Nayana Gaur I do not recommend using 96 well plates, and would suggest 6-well and 12-well plates instead, since they give more attachment space to the cells. Moreover, I had this experience of autologous serum and to be honest, with the best chances and culturing conditions you will not be able to obtain the quality you have while stimulating with hormones.
However, work on the purity of the cells after attachment. The less lymphocytes you have and the less RBCs and platelets you will have a better quality of differentiation and another trick: increase the serum concentration step by step and do not exceed 20%.