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. 2021 Nov 12;12:755846. doi: 10.3389/fimmu.2021.755846

Figure 2.

Figure 2

Flow cytometry analysis of monocyte subset on peripheral blood samples of a patient with ECD. (A) Flow cytometry analysis of monocyte subset in ECD remission. A.1 Monocyte population (green) is gated in dot plot SSC/CD45, and the zone is purified residual population (blue) by exclusion of T and NK lymphocytes with expression of CD7, granular cells with expression of CD16+ and CD14+) and B lymphocytes without CD16- but with expression of CD24+. A.2 The three subtypes of monocytes are gated with the combination CD16/CD14: classical monocytes are MO1 (black) CD16−CD14+ representing 92.9% of total monocytes. Intermediate monocytes are MO2 (blue) CD16+ CD14+low and represent 5.7% of total monocyte. Non-classical monocytes are MO3 (pink) expressing CD16+ without CD14 and representing 1.2% of total monocytes. A.3 In order to define the subtype, the gate is positioned with the help of all cells. (B) Flow cytometry analyses of monocyte subset in ECD flare The three monocytes subsets are gated with the combination CD16/CD14: classical monocytes are MO1 ((black) CD16− CD14+ representing 97.5% of total monocytes. Intermediate monocytes are MO2 (blue) CD16+ CD14+low representing 0.7% of total monocytes. Non classical monocytes are MO3 (pink) CD16+ CD14- representing 1% of total monocytes. CD45V500, CD7V450, CD16FITC, CD24PE, CD14APC-H7, and CD56PC7. BC, Beckman Coulter Immunotech, Miami, FL, USA; BD, BD Biosciences, San Jose, CA, USA; V500, Horizon V500; APC-H7, allophycocyanin-H7; PC7, phycoerythrin-cyanin-7; PE, phycoerythrin; V450, Horizon V450; FITC, fluorescein isothiocyanate. Peripheral blood mononuclear cells (PBMC) were stained with CD15, CD 14, CD13 PE-CF594, CD33, CD34, and CD45 KO, and monocytes subsets were sorted. PBMC were centrifugated on a microscope slide, dried at room temperature for 30 min, and stained using May–Gruenwald–Giemsa stain. Monocyte population is gated in dot plot SSC/CD45, and the zone is purified of residual population by exclusion of T and NK lymphocytes with expression of CD7, granular cells with expression of CD16+ and CD14+ and B lymphocytes without CD16− but with expression of CD24+.