研究目的
To establish an alternative automatic method for counting peripheral blood cells in Xenopus laevis and X. tropicalis using flow cytometry (FCM) with acridine orange (AO) staining, and to compare haematological phenotypes among different strains.
研究成果
The study successfully established a method for automatic counting and analysis of peripheral blood cells in Xenopus species using FCM with AO staining. This method allows for accurate differentiation of cell types and identification of haematological abnormalities, providing a valuable tool for research in genetically modified frogs and other species.
研究不足
The study acknowledges that higher order RNA structures may be stained as double-stranded nucleic acids, leading to potential false-positive results. Future studies are needed to devise methods for determining the exact amount of DNA and RNA without such interference.
1：Experimental Design and Method Selection:
The study utilized flow cytometry (FCM) with acridine orange (AO) staining to analyze and count peripheral blood cells in Xenopus laevis and X. tropicalis. The methodology was chosen for its ability to distinguish cell types based on size, presence of nucleus, and fluorescence intensity.
2：Sample Selection and Data Sources:
Blood samples were obtained from wild-type male African clawed frogs (X. laevis) and three inbred strains of X. tropicalis (Golden, Nigerian H, and Ivory Coast). Samples were prepared by cardiac puncture and treated with anti-coagulant EDTA-2Na.
3：List of Experimental Equipment and Materials:
Equipment included a FACS Aria II system for FCM analysis, a microplate reader for fluorescence measurement, and a haemocytometer for manual cell counting. Materials included acridine orange, Shaw’s diluting solution, and various staining reagents.
4：Experimental Procedures and Operational Workflow:
Blood cells were stained with AO and analyzed by FCM to distinguish cell types based on fluorescence intensity and light scattering properties. Manual counting with a haemocytometer was performed for comparison. Additional cytological analyses included MGG and PAS staining.
5：Data Analysis Methods:
Data were analyzed using FACS Diva 6.1 and FlowJo software. Statistical analyses were performed using SPSS, with differences between means analyzed using ANOVA and Tukey-Kramer tests.
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