so many previous studies just do one kind of cell (ex macrophage) of Immunofluorescence assays and confocal microscopy, compare the difference between the control and concentration of drug group of DAPI / antibody/ merge . here i want to do it in a direct adipocyte and macrophage co-culture cells. we know that DAPI stained nuclei both adipocyte and macrophage, then i use an antibody F4/80 (macrophage surface marker) to stain macrophage, can i use this assay to identify the infiltration of macrophage to adipocyte?
Hello
yes , you can use F4/80+CD11c+ population of ATMs , also in many of the studies observed increase in MCP-1 expression in adipose tissue contributes to the macrophage infiltration into this tissue , add to insulin resistance, and hepatic steatosis associated with obesity in mice.
for direct co-culture of macrophages and adipocytes In some experiments the macrophages were labeled by incubation for 5 min with PKH26 at a concentration of 1 µM mixed in Diluent B per manufacturer’s instructions. Cells were washed three times in PBS prior to selection on plastic by adhesion. Labeling efficiency was 100% as assessed by immunofluorescence microscopy. Macrophages were then passaged by scraping with 10 mM EDTA in PBS and added to differentiated adipocytes in 12-well dishes at concentrations of 10,000 and 100,000 cells per well. Macrophages were plated onto empty 12-well dishes as controls. Cells were grown in coculture for 48–72 h prior to analysis
see the attachment , i hope help you
Good Luck
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