(a–f, h, l–n) Inflamed skin was analysed 24 hr after a single topical application of 2.5 nM TPA and compared to healthy UT skin. (a) Recruitment of basophils to inflamed skin was analysed by FACS and presented as absolute numbers of basophils in ear skin following i.v. injection of PTX or vehicle control. Basophils were gated as CD45locKit–IgE+CD41+. (b) Expression of chemokines in whole skin and (c) expression of chemokine receptors in sorted skin basophils and mast cells were analysed by qRT-PCR and presented relative to the expression of the control gene cyclophilin. (d–f) Recruitment of basophils to inflamed skin was analysed as in panel (a): (d) after i.v. injection of 50 ng anti-CXCR2 antibody, (e) in Ccr2–/– mice, or (f) after i.v. injection of 50 ng anti-CXCR4 antibody. Suitable control populations were used to show the efficacy of a treatment where appropriate. (g, i–k) Basophils were isolated from the spleen of WT mice and (g) analysed for cell surface or intracellular expression of CXCR4 by flow cytometry. (h) Expression of IL-3 and TSLP in whole inflamed and untreated (UT) skin was analysed by qRT-PCR and presented relative to the expression of the control gene cyclophilin. (i) Splenic basophils were incubated with 1 μg/ml TSLP or media alone for 24 hr, and expression levels of CXCR4 transcripts were assessed by qRT-PCR. (j, k) Basophils were activated for 24 hr with 100 ng/ml IL-3, 1 μg/ml TSLP or isotype control, with PMA/ionomycin for 4 hr, or left UT before surface expression of CXCR4 was analysed by (j) FACS or (k) total CXCR4 expression (yellow) imaged with surface staining of CD45 (red) using Imagestream. (l–n) Recruitment of basophils to inflamed skin was analysed as in panel (a): (l) in Tcrb–/–, Tcrd–/– and Tcrbd–/– mice, (m) following intradermal (i.d.) injection of 20 ng anti-TSLP or anti-IL-3 (or both) antibodies into the TPA treated ear or (n) into the contralateral ear. Representative data are shown in panels (g, j [left panels]) as histograms of CXCR4 FACS stain and in panel (k) as images from Imagestream; all other data are expressed as means ± SEM. Statistical analyses were performed by one-way ANOVA multiple comparison (a–c, e, g, j, l–n) or two-tailed Student’s t-test (d, f, h–i); *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. UT = untreated; ns = not significant; BF = bright field.