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Blood, Vol. 112, Issue 13, 5026-5036, December 15, 2008
CX3CL1/fractalkine is released from apoptotic lymphocytes to stimulate macrophage chemotaxis
Blood Truman et al.
112: 5026
Supplemental materials for: Truman et al
Files in this Data Supplement:
- Figure S1. Immunoblotting using mouse anti-human FKN mAb (clone 81513) detects a 70 kDa band that is non-specific (JPG, 41.9 KB)
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The monoclonal anti-human FKN antibody (clone 81513) detects three major bands (90, 70, and 60 kDa) when used in Western blotting. The 70 kDa band was consistently found in samples prepared from apoptotic and non-apoptotic BL cells but the intensity of this band showed minimal variation even when cells were undergoing apoptosis suggesting it is non-specific. Indeed, despite being closely similar, this band was not identical in size to recombinant human FKN. Furthermore, this band was not detected in lysates prepared from mouse germinal B cells when immunoblotted using a polyclonal rabbit anti-mouse FKN antibody despite being detected using monoclonal anti-human FKN antibody (clone 81513). (A) Immunoblots of whole cell lysates showing changes in FKN following induction of apoptosis in BL2 cell lines. Blots were performed exactly as described in Fig. 2C except samples were run for longer in SDS PAGE to enhance separation of proteins. Samples were prepared at the indicated times following staurosporine-treatment and full-length recombinant human FKN (rFKN) that excluded the transmembrane and cytoplasmic domains of FKN was included as a positive control (50 ng/lane). (B) Immunoblots prepared using mouse anti-human FKN monoclonal Ab that cross-reacts with mouse FKN (clone 81513) (left panel) or rabbit anti-mouse FKN polyclonal Ab (Torrey Pines Biolabs) (right panel) of cell-free supernatants (equal volumes of TCA/acetone-precipitated supernatants per lane) showing changes in FKN in purified germinal centre B cells (GC) and follicular B cells (fol) from mouse spleens cultured in vitro for 2 hr. Both antibodies detect the 60 kDa form of FKN that is released by apoptotic cells.
- Figure S2. Expression of FKN and requirement of its receptor for macrophage recruitment in normal lymphoid tissue in situ (JPG, 109 KB)
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(A) Expression of FKN in normal lymphoid tissue in situ. Immunohistochemical staining was performed on splenic follicles of Balb/c mice immunized with SRBC to induce germinal centre reactions. DAB chromogen product (brown) represents FKN staining; hematoxylin counterstain. Primary anti-FKN mAb (clone 81513) (left panel); isotype control mAb (mouse IgG1) (right panel). GC, germinal centre of lymphoid follicle. Arrows indicate examples of tingible-body macrophages. Bar, 52 µm. Stained tissue was analyzed using a Zeiss Axioskop 2 microscope at 20°C with objective lens Plan-NEOFLUAR 20× (numerical aperture 0.50), and images were taken using Jenoptik ProgRes C14 camera with Openlab 4.0.2 software. (B) Expression of CD68 in normal lymphoid tissue in situ. Immunohistochemical staining was performed on splenic follicles of mice (wild-type Balb/c, CX3CR1−/− Balb/c or CD14−/− Balb/c) immunized with SRBC to induce germinal centre reactions. DAB chromogen product (brown) represents CD68 staining; hematoxylin counterstain. Primary biotinylated anti-CD68 Ab (clone FA-11). GC, germinal centre of lymphoid follicle, circled area represents region scored for follicular CD68 positive cells. Scoring of follicles: spleens of 3–6 animals of 3 different types (wild-type Balb/c, CX3CR1−/− Balb/c or CD14−/− Balb/c) were analyzed, and 10–50 follicles per spleen were scored by eye, using OpenLab software to determine the area of the follicle. The mean number of positive cells per µm2 of follicle for each mouse, and from this a value for each type of mouse (n=3–6) ± standard error, was calculated. Statistics were performed using Minitab with ANOVA. Bar, 106 µm. Stained tissue was analyzed using a Zeiss Axioskop 2 microscope at 20°C with objective lens ACHROPLAN 10× (numerical aperture 0.25) and images were taken using Jenoptik ProgRes C14 camera with Openlab 4.0.2 software.
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