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I. Rohwedder et al.
flow chamber using increasing shear forces. Adherent neutrophils were assessed for each shear stress level and related to the number of adherent neutrophils under low flow conditions (Figure 2F). Loss of SFK resulted in a decrease in shear stress resistant adhesion compared to
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wildtype cells. At wall shear stress levels of 140 dyn/cm2, only 36% of SFK-ko leukocytes could adhere to the flow chamber, while 84% of wildtype leukocytes were still attached. To analyze if these findings also apply under in vivo conditions, we correlated in vivo shear rates to
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chambers coated with Selectin/ICAM-1 and CXCL1. Time- lapse movies over 10 minutes were performed and leukocytes tracked for 200 time points. All data are present- ed as mean± standard error of the mean (SEM). *P<0.05; **P<0.005; ***P<0.001; n.s. : not significant. (unpaired Student t-test). (A and C) Representative single cell migration tracks (upper panel) and rose plots (lower panel) for crawling in vivo (A) and in vitro (C) are displayed. Red lines indicate migration in, black lines migration against flow direction. Arrows indicate the direction of flow. In rose diagrams, the area of each sector is proportional to the frequency of the migration vectors of tracked wildtype or SFK-ko neutrophils. At least 50 cells were analyzed for each strain. (B) Intraluminal crawling velocity. (D) Crawling velocity under flow in vitro. (E) Relative number of crawling cells per Field of View (FOV) in vitro using wild- type and SFK-ko neutrophils. (F) Neutrophil detachment was analyzed using whole blood from 4 wildtype and 4 SFK-ko mice in flow chambers coat- ed with E-Selectin/ICAM-1 and CXCL1. Flow was applied and increased every 30 seconds as indicated and numbers of attached cells per FOV counted. All data are presented as mean±SEM. *P<0.05; **P<0.005; *** P<0.001 (two-way ANOVA, Sidak multiple com- parison test). (G) In vivo correlation of shear rates and number of adherent cells in TNFα-stimulated cremaster muscle venules of 5 wildtype and 5 SFK-ko mice and 5 wildtype mice treat- ed for 3 hours with the tyrosine kinase inhibitor Dasatinib (10 mg/kg).
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Figure 2. Neutrophil shear stress resistance is impaired in the genetic absence of Src family kinases (SFK). In vivo crawling experiments were ana- lyzed in TNFα-stimulated cremaster muscle tissue of 5 wildtype and 5 SFK- knockout (ko) mice. Time-lapse movies over 15 minutes were performed and leukocytes were tracked for 15 min- utes. In vitro crawling experiments were analyzed using whole blood from 3 wildtype and 3 SFK-ko mice in flow
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haematologica | 2020; 105(7)