Figure 3.

KO MGLs have increased glutamate, and ROS release decreased phagocytosis but the globally similar inflammatory response to LPS as CTRL and KO^R MGL

(A) Glutamate release by MGLs. Bars represent mean ± SEM. Significance was tested by one-way ANOVA with Tukey’s multiple comparison test (^∗p = 0.0329, two different KO lines, one rescue line and two independent CTRL MGL lines with two clones).

(B) Measurement of reactive oxygen species (ROS) released in the conditioned media by MGLs. One-way ANOVA with Tukey’s multiple comparison test was used to assess the significance (^∗∗∗∗p < 0.0001). Bars represent mean ± SEM. Two independent cell lines for CTRL and KO MGLs and one isogenic rescue line with three independent experiments per sample were used.

(C) Migration assays using transwell chambers using ATP or CXC3L1. Significance was tested by one-way ANOVA with Tukey’s multiple comparison test (^∗p = 0.0261), two independent cell lines for CTRL and KO MGLs, and one isogenic rescue line with three independent experiments per sample.

(D) Heatmap showing the means for each cytokine released in the conditioned media for a given genotype (blue to red, low to high concentration in the media in pg/mL), three independent CTRL and KO MGL lines and two isogenic rescue lines were used.

(E and F) MIP-1 alpha and GM-CSF released in the conditioned media by MGLs was measured, respectively (in pg/mL). Significance was tested by one-way ANOVA with Tukey’s multiple comparison test (^∗∗∗∗p < 0.0001 and ^∗p = 0.0179, three independent CTRL and KO MGL lines and two isogenic rescue lines were used). Bars represent mean ± SD.

(G) Bright-field images of CTRL, KO, and KO^R MGLs engulfing zymosan particles. Once engulfed, the zymosan particles fluoresce in red, scale bar 200 μm. Blue is a live nuclear stain (Nucleo Blue).

(H) Phagocytosis percentage of zymosan particles compared to CTRL MGLs. Significance was tested by one-way ANOVA with Tukey’s multiple comparison test (^∗∗∗∗p < 0.0001 and ^∗p = 0.0289, two different isogenic KO/KO^R pairs, at least four biological replicates each, two independent CTRL MGL lines with three clones). Bars represent mean ± SEM. Each dot represents one sample.

(I) Phagocytosis of pHrodo-conjugated brain organoid-derived synaptosomes-enriched fractions measured as red fluorescent area normalized to hour = 0 and to CTRL baseline phagocytosis using incucyte. Significance was tested by one-way ANOVA (^∗p < 0.001) using one control and one KO isogenic pair with 5 independent experiments each. Bars represent mean ± SEM. (^∗p = 0.0184 and ^∗p = 0.0269, respectively).