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. 2003 Aug;109(4):536–546. doi: 10.1046/j.1365-2567.2003.01686.x

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© 2003 Blackwell Publishing Ltd

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(a) Detection of CC chemokine receptor 3 (CCR3) and interleukin-5 receptor alpha-subunit (IL-5Rα) co-expression on CD34⁺ cells by flow cytometry. An enriched population of CD34⁺ cells (purity >85%) from bone marrow non-adherent mononuclear cells (NAMNC) was collected by a magnetic cell separation technique. Cells were stained with CD34–phycoerythrin (PE), anti-CCR3–fluorescein isothiocyanate (FITC) and anti-IL-5Rα–peridinin chlorophyll protein (PerCp). (A) CD34-positive staining events were gated (gate R1); (B) cells within R1 were assessed for co-expression of both CCR3 and IL-5Rα; (C) a 99·9% confidence limit was set for this rare cell type (>0·1% staining with isotype-control antibodies); (D) region statistics show that of the total number of CD34⁺ cells counted, 3·42% were CD34⁺ CCR3⁺, 0·45% were CD34⁺ IL-5Rα⁺ and 0·45% were CD34⁺ CCR3⁺ IL-5Rα⁺ (isotype-control values are expressed in parenthesis). The proportion of CD34⁺ CCR3⁺ IL-5Rα⁺ cells represented 11% of the CD34⁺ CCR3⁺ cells and 50% of the CD34⁺ IL-5Rα⁺ cells in this sample. (b) Co-localization of mRNA for CCR3 to CD34⁺ cells using sequential immunocytochemistry (ICC) and in situ hybridization (ISH) on cytospins of bone marrow-derived CD34⁺ cells from an atopic asthmatic subject. (A)–(D) Staining with Dig-labelled CCR3 sense (A, B) and antisense (C, D) riboprobes, at low (magnification × 400; a and c) and high (magnification × 1000; b and d) power. Some of the CCR3 mRNA⁺ cells (dark blue) are indicated (→). (E) A combination of ICC and ISH in eosinophils (positive control cells) was performed. Single EG2⁺ stained red is indicated (▵) and single ISH⁺ (CCR3 mRNA) are dark blue (▴). Double positive cells are dark red (→). (F) Cytospins of purified bone marrow CD34⁺ cell ICC showing CD34⁺ cells (→). (G) Single ISH of CCR3 mRNA⁺ cells (→). (H) Double ICC/ISH of CD34⁺/CCR3⁺ cells (→).

(a) Detection of CC chemokine receptor 3 (CCR3) and interleukin-5 receptor alpha-subunit (IL-5Rα) co-expression on CD34⁺ cells by flow cytometry. An enriched population of CD34⁺ cells (purity >85%) from bone marrow non-adherent mononuclear cells (NAMNC) was collected by a magnetic cell separation technique. Cells were stained with CD34–phycoerythrin (PE), anti-CCR3–fluorescein isothiocyanate (FITC) and anti-IL-5Rα–peridinin chlorophyll protein (PerCp). (A) CD34-positive staining events were gated (gate R1); (B) cells within R1 were assessed for co-expression of both CCR3 and IL-5Rα; (C) a 99·9% confidence limit was set for this rare cell type (>0·1% staining with isotype-control antibodies); (D) region statistics show that of the total number of CD34⁺ cells counted, 3·42% were CD34⁺ CCR3⁺, 0·45% were CD34⁺ IL-5Rα⁺ and 0·45% were CD34⁺ CCR3⁺ IL-5Rα⁺ (isotype-control values are expressed in parenthesis). The proportion of CD34⁺ CCR3⁺ IL-5Rα⁺ cells represented 11% of the CD34⁺ CCR3⁺ cells and 50% of the CD34⁺ IL-5Rα⁺ cells in this sample. (b) Co-localization of mRNA for CCR3 to CD34⁺ cells using sequential immunocytochemistry (ICC) and in situ hybridization (ISH) on cytospins of bone marrow-derived CD34⁺ cells from an atopic asthmatic subject. (A)–(D) Staining with Dig-labelled CCR3 sense (A, B) and antisense (C, D) riboprobes, at low (magnification × 400; a and c) and high (magnification × 1000; b and d) power. Some of the CCR3 mRNA⁺ cells (dark blue) are indicated (→). (E) A combination of ICC and ISH in eosinophils (positive control cells) was performed. Single EG2⁺ stained red is indicated (▵) and single ISH⁺ (CCR3 mRNA) are dark blue (▴). Double positive cells are dark red (→). (F) Cytospins of purified bone marrow CD34⁺ cell ICC showing CD34⁺ cells (→). (G) Single ISH of CCR3 mRNA⁺ cells (→). (H) Double ICC/ISH of CD34⁺/CCR3⁺ cells (→).