![Figure 1. Purification of human monocyte subsets. (A) PBMCs were isolated by Ficoll-Paque and stained with anti-CD86, anti-CD14, and anti-CD16; CD86-positive cells (monocytes) are red, whereas CD86-negative (nonmonocytic) cells are black (i). NB: Percentages refer to CD86-positive monocyte subsets among all PBMCs, excluding CD86-negative cells (eg, CD16-positive NK cells and neutrophils) which protrude into the CD14+CD16++ monocyte gate in this dot plot. (B) After depletion of NK cells and neutrophils (CD16-positive nonmonocytic cells) using CD56 and CD15 MicroBeads (not shown), negatively isolated cells were separated into CD14++ (i) and CD14+/− cells (ii) using FITC-conjugated anti-CD14 Ab and accordingly anti-FITC MultiSort MicroBeads. (C) Both fractions were incubated with CD16 MicroBeads to separate CD14++ cells into CD14++CD16− (i) and CD14++CD16+ monocytes (ii), and to purify CD14+CD16++ monocytes (iii) from CD14+/− cells. Top line: Flow cytometric analysis; bottom line: microscopic images (Keyence BZ-8000J [Keyence Deutschland] equipped with a Plan Apo 60×/1.40 oil objective lens [Nikon], magnification 30×, room temperature) after cytospin and May-Grünwald-Giemsa staining. Representative examples from 12 independent experiments are shown. In each dot plot, subset-specific percentages of monocytic cells among total cells are shown as means ± SD.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/118/12/10.1182_blood-2011-01-326827/4/zh89991178390001.jpeg?Expires=1750168068&Signature=p6ej6QdKxFxjkjxvkbpwdq6numOtEby5eHrL2DjaZYQxKzdW7ehbdCf82dTpNzqxMspkzUccKGyM7qMykd3n0fCi4ZPoFLF73h7WQ8gB6PiKRqty2nXh50N9AMg6ZOfQnB9r8OY-FU243-Wlf5LSB48W536gz13xuMR1GJ9rqKO3ZY-P45V7MuXvA67GnSKhUeUUC3ftYSqpcLd139fiFCYxHxA0eduz~4kNJbYwaEKqyzk0CJo6Dt1iOLS~qWJwg8EASjLjyYgi3rx~86kv8zSMB0nB9LmHKMC5WmLwxoPHX6sbPe9VAPFvP-yaCKFZYsoi9HjRMpo6V~Mzg~fVgQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Purificationof human monocyte subsets. (A) PBMCs were isolated by Ficoll-Paque and stained with anti-CD86, anti-CD14, and anti-CD16; CD86-positive cells (monocytes) are red, whereas CD86-negative (nonmonocytic) cells are black (i). NB: Percentages refer to CD86-positive monocyte subsets among all PBMCs, excluding CD86-negative cells (eg, CD16-positive NK cells and neutrophils) which protrude into the CD14+CD16++ monocyte gate in this dot plot. (B) After depletion of NK cells and neutrophils (CD16-positive nonmonocytic cells) using CD56 and CD15 MicroBeads (not shown), negatively isolated cells were separated into CD14++ (i) and CD14+/− cells (ii) using FITC-conjugated anti-CD14 Ab and accordingly anti-FITC MultiSort MicroBeads. (C) Both fractions were incubated with CD16 MicroBeads to separate CD14++ cells into CD14++CD16− (i) and CD14++CD16+ monocytes (ii), and to purify CD14+CD16++ monocytes (iii) from CD14+/− cells. Top line: Flow cytometric analysis; bottom line: microscopic images (Keyence BZ-8000J [Keyence Deutschland] equipped with a Plan Apo 60×/1.40 oil objective lens [Nikon], magnification 30×, room temperature) after cytospin and May-Grünwald-Giemsa staining. Representative examples from 12 independent experiments are shown. In each dot plot, subset-specific percentages of monocytic cells among total cells are shown as means ± SD.
