Objective To investigate the role of intracellular Ca2+ and MERTK in the phagocytosis of human retinal pigment epithelial (RPE) cells, and reveal the relationship between MERTK and intracellular Ca2+. Methods The cultured RPE cells were incubated with rod outer segments (ROS) at 37℃, the phagocytosis was terminated at different incubation time points. The concentration of intracellular Ca2+ was assayed by Fluro-3/AM loading methods combined with fluorescence microscope and CCD system, and the mRNA level of MERTK gene was measured by reverse transcription polymerase chain reaction (RT-PCR). Treating the RPE cells with stimulator (A23187) or inhibitor (verapamile) of intracellular Ca2+ to observe the changes of MERTK gene expression. Results ROS adhered to hRPE cells at the 15th minute, and the ingestion saturated at the 24th hour. The concentration of intracellular Ca2+ increased at the 15th minute, and kept the high level in 24 hours. The level of MERTK mRNA increased at the 5th minute, and kept the high level duration the whole incubation. When RPE cells were treated by A23187, the expression of MERTK increased in a dose-dependent manner. After RPE cells was pretreated by A23187, the expression level of MERTK was higher in the proceeding incubation groups than which in the control group except at the 3rd hour. When RPE cells were treated by verapamil, the expression level of MERTK decreased in a dosedependent manner. After RPE cells were pretreated by verapamil , the expression level of MERTK was lower in all the proceeding incubation groups than which in the control group (Plt;0.05). Conclusion MERTK gene and Ca2+ play an important role in sustaining RPE cells phagocytizing ROS. As an up-stream regulator, the receptor tyrosine kinase MERTK keeps RPE cells phagocytizing ROS by starting the intracellular Ca2+.
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.