An investigation into how fixation and permeabilization affect antigens'mean fluorescence values and percentage of positive cells obtained through flow cytometry
10.3969/j.issn.1000-484X.2025.08.034
- VernacularTitle:固定破膜对流式细胞仪检测抗体平均荧光值和阳性细胞比例的影响研究
- Author:
Xianing GUO
1
;
Yuxin LIU
;
Song YU
;
Rong LI
;
Ye YANG
;
Jiaqing LI
;
Meng CAO
;
Jing LUAN
Author Information
1. 陈志南院士工作站,西安医学院基础与转化医学研究所,西安 710021
- Publication Type:Journal Article
- Keywords:
Flow cytometry;
Fixation and permeabilization;
Mean fluorescence intensity;
Positive cell proportion
- From:
Chinese Journal of Immunology
2025;41(8):1997-2002
- CountryChina
- Language:Chinese
-
Abstract:
Objective:Conventional myeloid cell staining methodologies were employed to stain splenocytes and peripheral blood cells by fixation and permeabilization and non-fixation and permeabilization methods,respectively,and effects of staining were compared following flow cytometry detection.Methods:Peripheral blood and spleen of three 8-week-old C57 male mice were divided into fixation and permeabilization group and non-fixation and permeabilization group for staining,and flow cytometry was used to detect mean fluorescence intensity(MFI)of each fluorescent antibody and proportion of positive cells.Results:In mouse peripheral blood samples,MFI of 7-AAD-PerCP-Cyanine5.5 and CD147-PE were lower in fixation and permeabilization group than non-fixation and permeabilization group,MFI of F4/80-FITC,MHC Class Ⅱ(I-A/I-E)-APC-R700,Ly-6c-APC-Cy7,CD206-BV421,CD45-BV510,CD11b-BV605,CD11c-BV650 and CD86-BV786 were higher than non-fixation and permeabilization group.Proportions of each immune cell in fixation and permeabilization group and non-fixation and permeabilization group were highly similar.In mouse spleen samples,MFI of antibodies in fixation and permeabilization group were higher than those in non-fixation and permeabilization group,with exception of CD147-PE,which had a lower MFI than non-fixation and permeabilization group;proportions of dendritic cells and Mon/Ly-6clow cells were lower than non-fixation and permeabilization group,whereas proportions of rest myeloid subpopula-tions were higher than non-fixation and permeabilization group.Conclusion:Fixation and permeabilization in peripheral blood cells can improve antibody MFI,but it has little effect on proportion of positive cells.Fixation and permeabilization can enhance antibody MFI in splenocytes,while effect on proportion of each positive cell is more significant.It is advised that when designing staining strate-gies,researchers attempt to choose alternative cell surface antigens for assay or optimize protocols through required pre-experiments in order to acquire stable and dependable results.
