Fig 5. Adhesion of RNA silenced macrophages.
MDMs were differentiated under conditions where CD11b or CD11c expression was downregulated by receptor specific siRNA. Control cells were treated with negative control siRNA. (A) Kinetic curves of adhering cells was recorded by EPIC BT measurement. Change in refractive index and thereby detected wavelength (Δλ) is plotted against time in the case of CD11b (dashed line), CD11c (dotted line) or negative control (black line) silenced MDMs of the same donor. The shaded area around each line shows the deviation between the parallel samples. One representative measurement out of three independent is shown. (B) Average+/-SD Δλ of three independent measurements was determined at the 30th minute of analysis. Paired t-test was used to compare the effect of CD11b or CD11c silencing compared to control siRNA treated samples. CD11c silencing was found to decrease Δλ significantly (p<0,5) (C) Cells were let to adhere for 30min at 37°C 5%CO2 on plates coated previously with 10μg/ml fibrinogen and blocked with PLL-g-PEG. Filamentous actin was stained with phalloidine-Alexa488 and contact zones were scanned for 200 cells with Olympus IX81 confocal microscope using 60x objective. Pictures were analysed for the amount of strong and weak actin clusters with ImageJ. CD11b and CD11c silenced cells were compared to negative control siRNA treated cells. MDMs with reduced CD11b had significantly more strong clusters and significantly less weak clusters compared to CD11c silenced cells. Results shown are mean +/- SD of three independent experiments, repeated measures ANOVA with Bonferroni post-test was used, * = p<0,05, ** = p<0,01. (D) The number of cells adhering with a given force was determined by the computer controlled micropipette. MDMs with silenced CD11b had significantly more cells that adhered with strong force compared to negative control siRNA treated cells. Differences in the case of CD11c silencing were not significant. Results shown are mean +/- SD of three independent experiments, repeated measures ANOVA with Bonferroni post-test was used, ** = p<0,01, *** = p<0,001.