Figure 5.

rHIgM22-mediated phagocytosis of myelin requires activity of the IgM Fc domain. (a,b) BV-2 cells were serum starved and IgMs were pre-incubated with Fc5µ antibody on ice for 30 minutes before treatment of cells with pHrodo-labeled myelin and IgMs. pHrodo signal was monitored on IncuCyte ZOOM for 24 hours. (a) Representative images of BV-2 cells treated with vehicle or IgMs (80 µg/mL) in the presence or absence of Fc5µ antibody at 24 hours after treatment. (b) Quantification of pHrodo uptake over 24 hours represented as area under the curve. The line graph shows the mean ± S.E.M. from 6 independent experiments. Statistical differences were calculated using 2-way ANOVA followed by Bonferroni posttests for rHIgM22 and isotype Ctrl IgM comparison (blue asterisks) and linear regression analysis for comparison to vehicle (details provided in Supplementary Fig. 5). (c) BV-2 cells were serum starved and IgMs were pre-incubated with Fc5µ antibody on ice for 30 minutes before treatment of cells with myelin and IgMs for 2 hours. The cells were lysed and analyzed for internalized CNPase by capillary immunoblot. Full blots are provided in Supplementary Fig. 6f,g and Supplementary Fig. 7a,b. (d) The line graph shows mean ± S.E.M. of CNPase/GAPDH ratio from 3 independent experiments. Statistical differences were calculated using 2-way ANOVA followed by Bonferroni post-tests. Black asterisks indicate significant difference compared to Vehicle treated cells. Blue asterisks indicate significant difference compared to cells treated with Isotype Ctrl at the corresponding concentration. Red asterisks indicate significant difference compared to cells treated with rHIgM22 at the corresponding concentration (***p < 0.001; **p < 0.01).