研究目的
To develop and use imaging flow cytometry and confocal microscopy-based assays to examine F-actin and phosphoinositide dynamics during different IpLITR-mediated phagocytic pathways.
研究成果
The study demonstrated that IpLITRs induce distinct phagocytic pathways characterized by unique F-actin and phosphoinositide dynamics. The ITAM-dependent pathway promotes canonical changes, while the ITAM-independent pathway induces alternative patterns. Both pathways result in target internalization into PI(3)P-enriched phagosomes, indicating a maturing phagosome compartment. This imaging-based platform is useful for further studies on immunoregulatory receptor-mediated phagocytosis.
研究不足
The study is limited by the use of a heterologous cell system (AD293 cells) which may not fully replicate the native immune cell environment. Additionally, the static nature of confocal microscopy images may not fully capture the dynamic processes of phagocytosis.
1:Experimental Design and Method Selection:
The study utilized imaging flow cytometry and confocal microscopy to examine F-actin and phosphoinositide dynamics during IpLITR-mediated phagocytosis.
2:Sample Selection and Data Sources:
AD293 cells stably expressing IpLITRs were used, with targets being αHA mAb or IgG3-coated microspheres.
3:List of Experimental Equipment and Materials:
Equipment included ImageStream X Mark II imaging flow cytometer and Zeiss 710 laser-scanning confocal microscope. Materials included LifeAct-GFP, PH-PLCδ1-GFP, and 2xFYVE-GFP probes.
4:Experimental Procedures and Operational Workflow:
Cells were transfected with GFP-tagged probes, incubated with target beads, and analyzed for phagocytic activity and molecular dynamics.
5:Data Analysis Methods:
Data were analyzed using imaging flow cytometry and confocal microscopy to assess phagocytic activity and the distribution of signaling molecules.
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