ObjectiveTo establish a methodology for alveolar macrophages (AMs) phagocytosis of AlexaFluor 488 (AF488) labeled bacteria by flow cytometry.MethodsStaphylococcus aureus and Streptococcus pneumoniae were labeled with different concentrations of AF488. A flow cytometric assay was used to quantify in vivo bacterial uptake by AMs. AMs and different ratio of fluorescent-labeled bacteria were incubated at 37 ℃ for 2 hours, 4 hours, 6 hours and 8 hours, respectively. AMs were washed with DPBS and extracellular fluorescence was quenched with 1% (w/v) trypan blue. Trypan blue was aspirated and phagocytosis of fluorescent-labeled bacteria by AMs was measured using a flow cytometry. Confocal microscopy was performed to ensure that bacterial in positive AM had been internalized rather than bound to the cell surface.ResultsWhen the concentration of AF488 was more than 50 μg/mL, the labeling rates of Staphylococcus aureus and Streptococcus pneumoniae were higher than 92% (P<0.05), and has quickly reached the upper limit. With the prolongation of incubation time, the phagocytic rate of AMs increased from 20.4% at 2 hours to 76.5% at 8 hours. With the increase in the number of bacteria, the phagocytic rate of AMs increased from 7.7% by ratio of 1∶10 to 85.1% by ratio of 1∶300.ConclusionDetection of AMs phagocytosis of AF488 labeled bacteria by flow cytometry is an effective method, but the dye concentration, incubation time and the proportion of bacteria will influence the results.
ObjectiveTo establisht a gut microbiota mice model for chronic obstructive pulmonary disease (COPD) with fecal microbiota transplantation (FMT) and its evaluation.MethodsThe mice received FMT from healthy individuals, COPD Ⅰ-Ⅱ subjects, or COPD Ⅲ–Ⅳ subjects. After microbiota depletion, the FMT was performed by a single oral administration of 100 μL per mouse every other day, for a total of 14 times in 28 days. On the 29th day, the peripheral blood mononuclear cells were analyzed, the gut microbiota of mice before and after FMT was analyzed by 16S rRNA sequencing, and the mice model were evaluated.ResultsThe operational taxonomic units, Chao 1 and Shannon indexes of mice all decreased significantly after antibiotic treatment (P<0.001), but increased significantly after FMT from healthy individuals, COPD Ⅰ-Ⅱ subjects, or COPD Ⅲ–Ⅳ subjects (P<0.05 or P<0.01). The abundance of Firmicutes, Proteobacteria and Actinobacteria in the guts of the mice in the healthy human FMT group, COPD Ⅰ-Ⅱ FMT group and COPD Ⅲ-Ⅳ FMT group were significantly different from those of the control group who only received phosphate buffer saline instead of FMT (P<0.05 or P<0.01). The auxiliary T lymphocytes and cytotoxic T lymphocytes were higher, but B lymphocytes decreased in the peripheral blood of the mice in the COPD Ⅰ-Ⅱ FMT group and COPD Ⅲ-Ⅳ FMT group (P<0.05 or P<0.01).ConclusionFMT can successfully establish a COPD gut microbiota research model.