5T4 Regulates CXCR4 Chemotaxis Figure 10. Influence of 5T4 on the chemokine receptors CXCR6 and CXCR3 in ES cells. (A), The expression and cellular localization of 5T4 and CXCR6 molecules on undifferenetiated and differentiating WT-ES cells was determined. Both molecules can be detected at the cell surface in differentiating cells with some areas of co-localization (5T4 = green; CXCR6 = red; composite, co-localisation = yellow; co-localized areas shown in separate channel). (B), Undifferentiated (white columns) and differentiating (black columns) ES cells were placed in a gradient of chemokine CXCL16 or not. (C), Immunofluorescence detection of 5T4 and CXCR3 in differentiating WT and 5T4KO-ES cells (5T4 = green, CXCR4 = red). Cell surface expression of 5T4 is present only on differentiating WT-ES cells whilst cell surface expression of CXCR3 is evident in both undifferentiated and differentiating WT and KO ES cells. (D), Undifferentiated WT (white columns) and 5T4KO (black columns) ES cells exhibit no CXCL10 dependent chemotaxis but differentiating WT and 5T4KO-ES cells, acquire significant chemotaxis towards CXCL10. doi:10.1371/journal.pone.0009982.g010 (FCS; Biosera), 2 mM L-glutamine, 100 units/ml penicillin and 100 mg/ml streptomycin solution and maintained at 37uC in a humidified atmosphere of 5% CO2/95% air. The hybridoma supernatant was clarified by centrifugation and proteins concen- trated by precipitation with 45% saturated ammonium sulphate, dialysed extensively against PBS and antibodies purified on protein G chromatography (HiTrap protein G column, GE Lifesciences; [58]). The antibodies eluted with 100 mM glycine (pH 2.5) into 1 M Tris salt (pH 9) were dialysed against PBS and used for subsequent assays. Characterization of m5T4 specific monoclonal antibodies Monoclonal antibodies were epitope mapped by FACS analysis performed against A9 cell lines bearing variant constructs of 5T4; m5T4, m/h5T4 or h/m5T4 chimeric constructs [59]. These cell lines were transfected with pCMVa neo constructs bearing either the full length m5T4, LRR1 of murine 5T4 fused to LRR2 of human 5T4 or LRR1 of human 5T4 fused to LRR2 of murine 5T4 respectively and maintained with 1mg/ml G418 selection (Sigma). Cells were suspended in FACS buffer (PBS, 0.2% bovine serum albumin, 0.1% sodium azide) and labelled with the appropriate concentration of anti-m5T4 monoclonal antibodies diluted in FACS buffer for 30 mins on ice, washed with FACS buffer and labelled with rabbit anti-mouse IgG conjugated to FITC (Dako, 1:40). 10,000 events were acquired using a Becton Dickinson FACScan and the data obtained was analysed using WinMIDI (version 2.8) software. Anti-m5T4 monoclonal antibodies were titrated by doubling dilution between 0 and 0.1 mg/ml by sandwich ELISA. 96-well ELISA plates (Falcon) were pre-coated at 4uC overnight with 1 mg/ml m5T4-pIgFc [58] in borate buffer (100 mM boric acid 150 mM NaCl; pH 8.5). All subsequent steps were performed at 37uC for 1 hour and the plates were washed three times with PBS containing 0.05% Tween 20 (Sigma; PBST) between each step. Non-specific binding was blocked using 2% low fat dried milk (marvel) in PBST (blocking buffer) at 37uC for 2 hours. Antibody binding was detected using goat anti-mouse IgG conjugated to HRP (Sigma, 1:1000) in blocking buffer. The plates were Figure 11. 5T4 is not required for CXCR7 surface expression. Immunofluorescence detection of 5T4 and CXCR7 in undifferentiated and differentiating WT and 5T4KO-ES cells (5T4 = green, CXCR7 = red). Cell surface expression of CXCR7 is high in undifferentiated ES cells (either WT or 5T4KO). In 3 day differentiating WT-ES cells, CXCR7 is relatively downregulated from the cell surface whereas in differentiating 5T4KO-ES surface CXCR7 is retained. doi:10.1371/journal.pone.0009982.g011 PLoS ONE | www.plosone.org 16 April 2010 | Volume 5 | Issue 4 | e9982