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( A ) Representative still images from live cell imaging sequences following a 2D scratch assay of WT and Sarm1-/- stimulated Mɸ. t=0 is immediately after the scratch and t=48 hours is 48 hours post-scratch. Scratch boundaries are indicated by yellow dashed lines. Scale bar = 300µm. ( B,C ) Quantification of the relative wound density of WT ( B ) or Sarm1-/- ( C ) Mɸ cultures after the scratch assay in A represented as a percentage of t=0. N=3 biological replicates. Error = SEM. *p<0.05; **p<0.01; ***p<0.005; letters (a,b,c) p<0.05. by two-way repeated measures ANOVA with Dunnett post-hoc test. Blue asterisk = mCSF compared to t=0. Green asterisk = IL-4 compared to t=0. Orange asterisk = LPS compared to t=0. a = mCSF compared to IL-4; b = mCSF compared to LPS; c = mCSF compared to IFNɣ conditions at indicated timepoints. ( D ) Representative still images from live cell imaging sequences following a 3D invasion assay in Matrigel of WT and Sarm1-/- stimulated Mɸ. t=0 immediately after the scratch and t=48 hours is 48 hours post-scratch. Scratch boundaries are indicated by yellow dashed lines. Scale bar = 300µm. ( E ) Quantification of the relative wound density of the invasion assay without <t>C5a</t> added (top) and with C5a added as a chemoattractant (bottom) represented as a percentage of t=0. N=2-4 biological replicates. Error = SEM. Letters (b,c) p<0.05; **p<0.01; ***p<0.005; ****p<0.001 by two-way ANOVA, main effects model with Tukey post-hoc test after simple linear regression model indicated slopes were not different. Wildtype Mɸ + C5a main effects could not be calculated because slopes were significantly different. Orange asterisk = LPS compared to t=0. b = mCSF compared to LPS; c = mCSF compared to IFNɣ conditions at indicated timepoints. ( F ) Representative images of F4/80 (green) positive Mɸ stained for Oil Red O lipids (magenta) following a myelin challenge assay. No clearance indicates a 24 hour myelin treatment and clearance indicates 24 hours of myelin treatment followed by myelin removal for 24 hours. Scale bar = 100µm. ( G ) Quantification of OilRedO intensity per Mɸ (normalized to Mɸ cell area). N = 3 biological replicates and each data point = 1 Mɸ. Mean +/- SEM is shown. Outliers were removed with ROUT Q=1. **p<0.01; ***p<0.005; ****p<0.001 by Kruskal-Wallis test with Dunn’s correction for multiple comparisons. Black asterisk = WT to Sarm1-/- comparisons. Purple asterisk = Sarm1-/- to Sarm1-/- comparisons. Blue asterisk = WT to WT comparisons.
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( A ) Representative still images from live cell imaging sequences following a 2D scratch assay of WT and Sarm1-/- stimulated Mɸ. t=0 is immediately after the scratch and t=48 hours is 48 hours post-scratch. Scratch boundaries are indicated by yellow dashed lines. Scale bar = 300µm. ( B,C ) Quantification of the relative wound density of WT ( B ) or Sarm1-/- ( C ) Mɸ cultures after the scratch assay in A represented as a percentage of t=0. N=3 biological replicates. Error = SEM. *p<0.05; **p<0.01; ***p<0.005; letters (a,b,c) p<0.05. by two-way repeated measures ANOVA with Dunnett post-hoc test. Blue asterisk = mCSF compared to t=0. Green asterisk = IL-4 compared to t=0. Orange asterisk = LPS compared to t=0. a = mCSF compared to IL-4; b = mCSF compared to LPS; c = mCSF compared to IFNɣ conditions at indicated timepoints. ( D ) Representative still images from live cell imaging sequences following a 3D invasion assay in Matrigel of WT and Sarm1-/- stimulated Mɸ. t=0 immediately after the scratch and t=48 hours is 48 hours post-scratch. Scratch boundaries are indicated by yellow dashed lines. Scale bar = 300µm. ( E ) Quantification of the relative wound density of the invasion assay without C5a added (top) and with C5a added as a chemoattractant (bottom) represented as a percentage of t=0. N=2-4 biological replicates. Error = SEM. Letters (b,c) p<0.05; **p<0.01; ***p<0.005; ****p<0.001 by two-way ANOVA, main effects model with Tukey post-hoc test after simple linear regression model indicated slopes were not different. Wildtype Mɸ + C5a main effects could not be calculated because slopes were significantly different. Orange asterisk = LPS compared to t=0. b = mCSF compared to LPS; c = mCSF compared to IFNɣ conditions at indicated timepoints. ( F ) Representative images of F4/80 (green) positive Mɸ stained for Oil Red O lipids (magenta) following a myelin challenge assay. No clearance indicates a 24 hour myelin treatment and clearance indicates 24 hours of myelin treatment followed by myelin removal for 24 hours. Scale bar = 100µm. ( G ) Quantification of OilRedO intensity per Mɸ (normalized to Mɸ cell area). N = 3 biological replicates and each data point = 1 Mɸ. Mean +/- SEM is shown. Outliers were removed with ROUT Q=1. **p<0.01; ***p<0.005; ****p<0.001 by Kruskal-Wallis test with Dunn’s correction for multiple comparisons. Black asterisk = WT to Sarm1-/- comparisons. Purple asterisk = Sarm1-/- to Sarm1-/- comparisons. Blue asterisk = WT to WT comparisons.

Journal: bioRxiv

Article Title: SARM1 is required for macrophage immunophenotype switching that is essential for nerve repair

doi: 10.64898/2026.04.07.716973

Figure Lengend Snippet: ( A ) Representative still images from live cell imaging sequences following a 2D scratch assay of WT and Sarm1-/- stimulated Mɸ. t=0 is immediately after the scratch and t=48 hours is 48 hours post-scratch. Scratch boundaries are indicated by yellow dashed lines. Scale bar = 300µm. ( B,C ) Quantification of the relative wound density of WT ( B ) or Sarm1-/- ( C ) Mɸ cultures after the scratch assay in A represented as a percentage of t=0. N=3 biological replicates. Error = SEM. *p<0.05; **p<0.01; ***p<0.005; letters (a,b,c) p<0.05. by two-way repeated measures ANOVA with Dunnett post-hoc test. Blue asterisk = mCSF compared to t=0. Green asterisk = IL-4 compared to t=0. Orange asterisk = LPS compared to t=0. a = mCSF compared to IL-4; b = mCSF compared to LPS; c = mCSF compared to IFNɣ conditions at indicated timepoints. ( D ) Representative still images from live cell imaging sequences following a 3D invasion assay in Matrigel of WT and Sarm1-/- stimulated Mɸ. t=0 immediately after the scratch and t=48 hours is 48 hours post-scratch. Scratch boundaries are indicated by yellow dashed lines. Scale bar = 300µm. ( E ) Quantification of the relative wound density of the invasion assay without C5a added (top) and with C5a added as a chemoattractant (bottom) represented as a percentage of t=0. N=2-4 biological replicates. Error = SEM. Letters (b,c) p<0.05; **p<0.01; ***p<0.005; ****p<0.001 by two-way ANOVA, main effects model with Tukey post-hoc test after simple linear regression model indicated slopes were not different. Wildtype Mɸ + C5a main effects could not be calculated because slopes were significantly different. Orange asterisk = LPS compared to t=0. b = mCSF compared to LPS; c = mCSF compared to IFNɣ conditions at indicated timepoints. ( F ) Representative images of F4/80 (green) positive Mɸ stained for Oil Red O lipids (magenta) following a myelin challenge assay. No clearance indicates a 24 hour myelin treatment and clearance indicates 24 hours of myelin treatment followed by myelin removal for 24 hours. Scale bar = 100µm. ( G ) Quantification of OilRedO intensity per Mɸ (normalized to Mɸ cell area). N = 3 biological replicates and each data point = 1 Mɸ. Mean +/- SEM is shown. Outliers were removed with ROUT Q=1. **p<0.01; ***p<0.005; ****p<0.001 by Kruskal-Wallis test with Dunn’s correction for multiple comparisons. Black asterisk = WT to Sarm1-/- comparisons. Purple asterisk = Sarm1-/- to Sarm1-/- comparisons. Blue asterisk = WT to WT comparisons.

Article Snippet: Additional media with or without recombinant mouse complement component C5a (R&D system, #2150-C5/CF) was added after incubation.

Techniques: Live Cell Imaging, Wound Healing Assay, Invasion Assay, Staining