Guang Yang, China
Chinese PLA general hospital Geriatric nephrologyPresenter of 1 Presentation
ESTABLISHMENT OF MACROPHAGE ISOLATION AND INDUCTION CULTURE AND EFFECTS OF HIGH GLUCOSE ON MACROPHAGE POLARIZATION
Abstract
Background and Aims
To establish a method for isolation and induction of mouse bone marrow-derived macrophages (BMDM) and to evaluate the effect of high glucose on macrophage polarization.
Methods
BMDMs were isolated for the femur and tibia of 6-8 weeks C57BL/6 mice, and identified by flow cytometry, then be divided into M1 (induced by LPS and IFN-γ), M2(induced by IL-4), normal glusose (5.6 mmol/l), high glucose (30 mmol/l) and osmotic pressure control group (normal glucose+mannitol 24.4mmol/L), stimulated by high glucose for 24 and 48 hours, collect cells for flow cytometry and qPCR detection of M1 and M2 cell surface marker expression.
Results
BMDMs were isolated successfully, the proportion of CD11b and F4/80 double positive cells was above 95%; After induction, In M1 subgroup, the flow cytometry results suggested that CD86 positive rate increased significantly, qPCR results showed that IL-1b, iNOS expression increased significantly; in M2 subgroup, CD206 positive rate increased significantly, the expression of Arg1 and Ym1 were significantly increased. After stimulation with high glucose, with the prolongation of high glucose stimulation time, the ratio of CD86 positive rate in BMDMs increased gradually, while the positive rate of CD206 did not increase significantly; qPCR results showed that with high glucose stimulation over time, M1 macrophage labeled IL-1b, iNOS expression was significantly increased (p <0.05).
Conclusions
We have successfully established a method for isolation and induction culture of BMDMs. After 48 hours of high glucose stimulation, macrophages will be polarized to M1.