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Fig. 7. Hyaluronan binding of human CD44s expressing NIH-3T3 cells after hyaluronidase treatment. (A) NIH-3T3 cells transfected with vector alone or human CD44s were detached from culture dishes, resuspended in DMEM plus 10% DCS and treated with S. hyalurolyticus hyaluronidase. After washing twice in PBS, cells were incubated either with FITC-hyaluronan (FITC-HA, green) or CD44-Fc (red). As controls, cells were also incubated with Endo180-Fc (grey) or medium alone (black). Fc chimaeras were detected with PE-anti-human Fc. (B) NIH-3T3 cells transfected with vector alone or human CD44s were cultured on coverslips overnight and then treated with type IV-S hyaluronidase. After washing, cells were stained with FITC-hyaluronan (green) and anti-human CD44 monoclonal antibody E1/2 (red) as described in Fig. 2. Nuclei were visualised with TO-PRO-3 (blue). To illustrate the variation in FITC-hyaluronan binding following hyaluronidase treatment, two separate fields of view are shown (lower two rows). (C) MDA-MB-468 and MDA-MB-231 cells were analysed by flow cytometry for FITC-HA binding following treatment with either medium alone (green) or treatment with S. hyalurolyticus hyaluronidase (dotted green). Black lines represent binding of secondary antibody alone. Bar, 100 µm.
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