Objective:To investigate the flow cytometry cell sorting regimen for M1 macrophages and the association between M1 mac-rophage polarization and nonalcoholic fatty liver disease(NAFLD).Methods:High-fat diet(HFD)was used to establish a mouse model of NAFLD.Twelve C57BL/6 mice were randomly divided into control group(normal diet)and HFD group using a random num-ber table and were fed for 24 weeks.Metabolic markers including blood glucose and blood lipids were measured;quantitative real-time PCR was used to measure the factors associated with M1 macrophages in mice;HE staining was used to observe liver pathology.The Percoll gradient centrifugation method was used to collect liver Kupffer cells,and flow cytometry was used to measure M1 macro-phages in mouse liver(sorting regimen:FSC-A/SSC-A for grouping and removing red blood cells and impurities in the liver;FITC CD45(+)/PE-cy7 CD11clow for grouping leukocytes;APC CD115(+)/Percp cy5.5 CD11bhigh for the screening of monocytes;Apc-cy7 F4/80low/PE Ly-6Chigh for separating M1 macrophages).Results:Com-pared with the control group at week 24,the HFD group had signifi-cant increases in the indicators of body weight[(28.35±1.71)g vs.(38.43±4.41)g,P<0.001),liver weight[(1.03±0.18)g vs.(1.85±0.41)g,P=0.003),fasting blood glucose[(10.23±1.58)mmol/L vs.(7.07±0.58)mmol/L,P＜0.001)],insulin[(18.62±3.84)pg/mL vs.(28.84±8.3)pg/mL,P＜0.001)],triglyceride[(2.97±0.67)mmol/L vs.(4.05±0.99)mmol/L,P=0.01)],cholesterol[(0.23±0.06)mmol/L vs.(0.55±0.17)mmol/L,P<0.001)],alanine aminotransferase[5.67(3.16,9.23)U/L vs.35.86(19.68,58.33)U/L,P=0.003],and aspartate aminotransferase[53.14(38.18,64.40)U/L vs.155.10(113.60,192.20)U/L,P<0.001],and there was a significant increase in M1 macrophage polarization in NAFLD mice[42.00%(26.50,45.50)vs.9.95%(3.1,12),P=0.003].There were significant increases in the mRNA levels of IL-β,IL-6,F4/80,and TNF-α in the liver of mice induced by HFD.Conclusion:The flow cytometry sorting regimen can be used to measure M1 macrophages in the liver.Significant aggravation of inflammatory response is observed in NAFLD,and M1 macrophage polarization is positively correlated with the onset of NAFLD.