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conducted imgl cell sequencing  (Novogene)
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(A) Phagocytosis of synaptosomes by WT or TREM2 KO <t>iMGL</t> treated with SEMA6D (5 μg/ml). y-axis shows total well red fluorescent I.I., integrated intensity. Data averaged from three independent experiments (shown in fig. S11 A ); mean ± SD values. (B) Quantification of phagocytosis of synaptosomes by WT or TREM2 KO iMGL treated with SEMA6D at 24 hr. Data represent the three independent experiments performed in triplicate; median ± interquartile values at 24 hrs, p-values obtained from using a linear mixed-effects model (shown in fig. S11 A ). (C) Quantification of TNF-α and IL-6 in media from WT or TREM2 KO iMGL treated with SEMA6D (10 μg/ml), using the Mesoscale V-plex neuroinflammation panel. Boxplots indicate median and interquartile ranges, p-values calculated using linear regression. (D) Representative western blots of p-SYK and total SYK in WT and TREM2 KO iMGL treated with SEMA6D (10 μg/ml). β-actin as loading control (E) Quantification of p-SYK/total SYK chemiluminescent western blot analysis, n=3 (individual replicates shown in fig. S12 ). (F) Bulk RNA-seq effect size distribution indicating the transcriptional changes induced by SEMA6D treatment in WT and TREM2 KO iMGL. (G) Transcriptional changes in the TREM2 crosstalk sub-network associated with SEMA6D treatment (WT or TREM2 KO vs. untreated control) or TREM2 KO (TREM2 KO vs. WT, no treatment). Only TREM2 sub-network genes differentially expressed in at least one comparison were included. (H) Transcriptional effects of the same conditions across clinically and biologically relevant gene signatures. Background corresponds to 500 randomly selected genes. Solid dots correspond to a 1% FDR significance threshold for comparing the effect size distribution to the corresponding background (Mann-Whitney test). (I) Transcriptional effects of the experimental conditions across a representative subset of highly differentially expressed genes from the signatures in (H). (J) Representative confocal immunofluorescence of 3D triculture model consisting of differentiated ADAD hNPCs (neuron/glia – GFP, green) and iMGLs (IBA1, red), stained for Aβ (blue). (K) Analysis of Aβ40 and Aβ42 expression in conditioned media of triculture model after 24 hours of pre-treatments followed by 3 hours of co-retreatments. Control IgG (n=5) and SEMA6D (n=5). Vertical bars; median. Unpaired two-tailed t-test.
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(A) Phagocytosis of synaptosomes by WT or TREM2 KO <t>iMGL</t> treated with SEMA6D (5 μg/ml). y-axis shows total well red fluorescent I.I., integrated intensity. Data averaged from three independent experiments (shown in fig. S11 A ); mean ± SD values. (B) Quantification of phagocytosis of synaptosomes by WT or TREM2 KO iMGL treated with SEMA6D at 24 hr. Data represent the three independent experiments performed in triplicate; median ± interquartile values at 24 hrs, p-values obtained from using a linear mixed-effects model (shown in fig. S11 A ). (C) Quantification of TNF-α and IL-6 in media from WT or TREM2 KO iMGL treated with SEMA6D (10 μg/ml), using the Mesoscale V-plex neuroinflammation panel. Boxplots indicate median and interquartile ranges, p-values calculated using linear regression. (D) Representative western blots of p-SYK and total SYK in WT and TREM2 KO iMGL treated with SEMA6D (10 μg/ml). β-actin as loading control (E) Quantification of p-SYK/total SYK chemiluminescent western blot analysis, n=3 (individual replicates shown in fig. S12 ). (F) Bulk RNA-seq effect size distribution indicating the transcriptional changes induced by SEMA6D treatment in WT and TREM2 KO iMGL. (G) Transcriptional changes in the TREM2 crosstalk sub-network associated with SEMA6D treatment (WT or TREM2 KO vs. untreated control) or TREM2 KO (TREM2 KO vs. WT, no treatment). Only TREM2 sub-network genes differentially expressed in at least one comparison were included. (H) Transcriptional effects of the same conditions across clinically and biologically relevant gene signatures. Background corresponds to 500 randomly selected genes. Solid dots correspond to a 1% FDR significance threshold for comparing the effect size distribution to the corresponding background (Mann-Whitney test). (I) Transcriptional effects of the experimental conditions across a representative subset of highly differentially expressed genes from the signatures in (H). (J) Representative confocal immunofluorescence of 3D triculture model consisting of differentiated ADAD hNPCs (neuron/glia – GFP, green) and iMGLs (IBA1, red), stained for Aβ (blue). (K) Analysis of Aβ40 and Aβ42 expression in conditioned media of triculture model after 24 hours of pre-treatments followed by 3 hours of co-retreatments. Control IgG (n=5) and SEMA6D (n=5). Vertical bars; median. Unpaired two-tailed t-test.
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(A) Phagocytosis of synaptosomes by WT or TREM2 KO <t>iMGL</t> treated with SEMA6D (5 μg/ml). y-axis shows total well red fluorescent I.I., integrated intensity. Data averaged from three independent experiments (shown in fig. S11 A ); mean ± SD values. (B) Quantification of phagocytosis of synaptosomes by WT or TREM2 KO iMGL treated with SEMA6D at 24 hr. Data represent the three independent experiments performed in triplicate; median ± interquartile values at 24 hrs, p-values obtained from using a linear mixed-effects model (shown in fig. S11 A ). (C) Quantification of TNF-α and IL-6 in media from WT or TREM2 KO iMGL treated with SEMA6D (10 μg/ml), using the Mesoscale V-plex neuroinflammation panel. Boxplots indicate median and interquartile ranges, p-values calculated using linear regression. (D) Representative western blots of p-SYK and total SYK in WT and TREM2 KO iMGL treated with SEMA6D (10 μg/ml). β-actin as loading control (E) Quantification of p-SYK/total SYK chemiluminescent western blot analysis, n=3 (individual replicates shown in fig. S12 ). (F) Bulk RNA-seq effect size distribution indicating the transcriptional changes induced by SEMA6D treatment in WT and TREM2 KO iMGL. (G) Transcriptional changes in the TREM2 crosstalk sub-network associated with SEMA6D treatment (WT or TREM2 KO vs. untreated control) or TREM2 KO (TREM2 KO vs. WT, no treatment). Only TREM2 sub-network genes differentially expressed in at least one comparison were included. (H) Transcriptional effects of the same conditions across clinically and biologically relevant gene signatures. Background corresponds to 500 randomly selected genes. Solid dots correspond to a 1% FDR significance threshold for comparing the effect size distribution to the corresponding background (Mann-Whitney test). (I) Transcriptional effects of the experimental conditions across a representative subset of highly differentially expressed genes from the signatures in (H). (J) Representative confocal immunofluorescence of 3D triculture model consisting of differentiated ADAD hNPCs (neuron/glia – GFP, green) and iMGLs (IBA1, red), stained for Aβ (blue). (K) Analysis of Aβ40 and Aβ42 expression in conditioned media of triculture model after 24 hours of pre-treatments followed by 3 hours of co-retreatments. Control IgG (n=5) and SEMA6D (n=5). Vertical bars; median. Unpaired two-tailed t-test.
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(A) Phagocytosis of synaptosomes by WT or TREM2 KO <t>iMGL</t> treated with SEMA6D (5 μg/ml). y-axis shows total well red fluorescent I.I., integrated intensity. Data averaged from three independent experiments (shown in fig. S11 A ); mean ± SD values. (B) Quantification of phagocytosis of synaptosomes by WT or TREM2 KO iMGL treated with SEMA6D at 24 hr. Data represent the three independent experiments performed in triplicate; median ± interquartile values at 24 hrs, p-values obtained from using a linear mixed-effects model (shown in fig. S11 A ). (C) Quantification of TNF-α and IL-6 in media from WT or TREM2 KO iMGL treated with SEMA6D (10 μg/ml), using the Mesoscale V-plex neuroinflammation panel. Boxplots indicate median and interquartile ranges, p-values calculated using linear regression. (D) Representative western blots of p-SYK and total SYK in WT and TREM2 KO iMGL treated with SEMA6D (10 μg/ml). β-actin as loading control (E) Quantification of p-SYK/total SYK chemiluminescent western blot analysis, n=3 (individual replicates shown in fig. S12 ). (F) Bulk RNA-seq effect size distribution indicating the transcriptional changes induced by SEMA6D treatment in WT and TREM2 KO iMGL. (G) Transcriptional changes in the TREM2 crosstalk sub-network associated with SEMA6D treatment (WT or TREM2 KO vs. untreated control) or TREM2 KO (TREM2 KO vs. WT, no treatment). Only TREM2 sub-network genes differentially expressed in at least one comparison were included. (H) Transcriptional effects of the same conditions across clinically and biologically relevant gene signatures. Background corresponds to 500 randomly selected genes. Solid dots correspond to a 1% FDR significance threshold for comparing the effect size distribution to the corresponding background (Mann-Whitney test). (I) Transcriptional effects of the experimental conditions across a representative subset of highly differentially expressed genes from the signatures in (H). (J) Representative confocal immunofluorescence of 3D triculture model consisting of differentiated ADAD hNPCs (neuron/glia – GFP, green) and iMGLs (IBA1, red), stained for Aβ (blue). (K) Analysis of Aβ40 and Aβ42 expression in conditioned media of triculture model after 24 hours of pre-treatments followed by 3 hours of co-retreatments. Control IgG (n=5) and SEMA6D (n=5). Vertical bars; median. Unpaired two-tailed t-test.
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conductivity cell orion 01310md  (Thermo Fisher)
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(A) Phagocytosis of synaptosomes by WT or TREM2 KO <t>iMGL</t> treated with SEMA6D (5 μg/ml). y-axis shows total well red fluorescent I.I., integrated intensity. Data averaged from three independent experiments (shown in fig. S11 A ); mean ± SD values. (B) Quantification of phagocytosis of synaptosomes by WT or TREM2 KO iMGL treated with SEMA6D at 24 hr. Data represent the three independent experiments performed in triplicate; median ± interquartile values at 24 hrs, p-values obtained from using a linear mixed-effects model (shown in fig. S11 A ). (C) Quantification of TNF-α and IL-6 in media from WT or TREM2 KO iMGL treated with SEMA6D (10 μg/ml), using the Mesoscale V-plex neuroinflammation panel. Boxplots indicate median and interquartile ranges, p-values calculated using linear regression. (D) Representative western blots of p-SYK and total SYK in WT and TREM2 KO iMGL treated with SEMA6D (10 μg/ml). β-actin as loading control (E) Quantification of p-SYK/total SYK chemiluminescent western blot analysis, n=3 (individual replicates shown in fig. S12 ). (F) Bulk RNA-seq effect size distribution indicating the transcriptional changes induced by SEMA6D treatment in WT and TREM2 KO iMGL. (G) Transcriptional changes in the TREM2 crosstalk sub-network associated with SEMA6D treatment (WT or TREM2 KO vs. untreated control) or TREM2 KO (TREM2 KO vs. WT, no treatment). Only TREM2 sub-network genes differentially expressed in at least one comparison were included. (H) Transcriptional effects of the same conditions across clinically and biologically relevant gene signatures. Background corresponds to 500 randomly selected genes. Solid dots correspond to a 1% FDR significance threshold for comparing the effect size distribution to the corresponding background (Mann-Whitney test). (I) Transcriptional effects of the experimental conditions across a representative subset of highly differentially expressed genes from the signatures in (H). (J) Representative confocal immunofluorescence of 3D triculture model consisting of differentiated ADAD hNPCs (neuron/glia – GFP, green) and iMGLs (IBA1, red), stained for Aβ (blue). (K) Analysis of Aβ40 and Aβ42 expression in conditioned media of triculture model after 24 hours of pre-treatments followed by 3 hours of co-retreatments. Control IgG (n=5) and SEMA6D (n=5). Vertical bars; median. Unpaired two-tailed t-test.
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(A) Phagocytosis of synaptosomes by WT or TREM2 KO <t>iMGL</t> treated with SEMA6D (5 μg/ml). y-axis shows total well red fluorescent I.I., integrated intensity. Data averaged from three independent experiments (shown in fig. S11 A ); mean ± SD values. (B) Quantification of phagocytosis of synaptosomes by WT or TREM2 KO iMGL treated with SEMA6D at 24 hr. Data represent the three independent experiments performed in triplicate; median ± interquartile values at 24 hrs, p-values obtained from using a linear mixed-effects model (shown in fig. S11 A ). (C) Quantification of TNF-α and IL-6 in media from WT or TREM2 KO iMGL treated with SEMA6D (10 μg/ml), using the Mesoscale V-plex neuroinflammation panel. Boxplots indicate median and interquartile ranges, p-values calculated using linear regression. (D) Representative western blots of p-SYK and total SYK in WT and TREM2 KO iMGL treated with SEMA6D (10 μg/ml). β-actin as loading control (E) Quantification of p-SYK/total SYK chemiluminescent western blot analysis, n=3 (individual replicates shown in fig. S12 ). (F) Bulk RNA-seq effect size distribution indicating the transcriptional changes induced by SEMA6D treatment in WT and TREM2 KO iMGL. (G) Transcriptional changes in the TREM2 crosstalk sub-network associated with SEMA6D treatment (WT or TREM2 KO vs. untreated control) or TREM2 KO (TREM2 KO vs. WT, no treatment). Only TREM2 sub-network genes differentially expressed in at least one comparison were included. (H) Transcriptional effects of the same conditions across clinically and biologically relevant gene signatures. Background corresponds to 500 randomly selected genes. Solid dots correspond to a 1% FDR significance threshold for comparing the effect size distribution to the corresponding background (Mann-Whitney test). (I) Transcriptional effects of the experimental conditions across a representative subset of highly differentially expressed genes from the signatures in (H). (J) Representative confocal immunofluorescence of 3D triculture model consisting of differentiated ADAD hNPCs (neuron/glia – GFP, green) and iMGLs (IBA1, red), stained for Aβ (blue). (K) Analysis of Aβ40 and Aβ42 expression in conditioned media of triculture model after 24 hours of pre-treatments followed by 3 hours of co-retreatments. Control IgG (n=5) and SEMA6D (n=5). Vertical bars; median. Unpaired two-tailed t-test.
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orion duraprobe 4-cell conductivity probe  (Thermo Fisher)
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(A) Phagocytosis of synaptosomes by WT or TREM2 KO <t>iMGL</t> treated with SEMA6D (5 μg/ml). y-axis shows total well red fluorescent I.I., integrated intensity. Data averaged from three independent experiments (shown in fig. S11 A ); mean ± SD values. (B) Quantification of phagocytosis of synaptosomes by WT or TREM2 KO iMGL treated with SEMA6D at 24 hr. Data represent the three independent experiments performed in triplicate; median ± interquartile values at 24 hrs, p-values obtained from using a linear mixed-effects model (shown in fig. S11 A ). (C) Quantification of TNF-α and IL-6 in media from WT or TREM2 KO iMGL treated with SEMA6D (10 μg/ml), using the Mesoscale V-plex neuroinflammation panel. Boxplots indicate median and interquartile ranges, p-values calculated using linear regression. (D) Representative western blots of p-SYK and total SYK in WT and TREM2 KO iMGL treated with SEMA6D (10 μg/ml). β-actin as loading control (E) Quantification of p-SYK/total SYK chemiluminescent western blot analysis, n=3 (individual replicates shown in fig. S12 ). (F) Bulk RNA-seq effect size distribution indicating the transcriptional changes induced by SEMA6D treatment in WT and TREM2 KO iMGL. (G) Transcriptional changes in the TREM2 crosstalk sub-network associated with SEMA6D treatment (WT or TREM2 KO vs. untreated control) or TREM2 KO (TREM2 KO vs. WT, no treatment). Only TREM2 sub-network genes differentially expressed in at least one comparison were included. (H) Transcriptional effects of the same conditions across clinically and biologically relevant gene signatures. Background corresponds to 500 randomly selected genes. Solid dots correspond to a 1% FDR significance threshold for comparing the effect size distribution to the corresponding background (Mann-Whitney test). (I) Transcriptional effects of the experimental conditions across a representative subset of highly differentially expressed genes from the signatures in (H). (J) Representative confocal immunofluorescence of 3D triculture model consisting of differentiated ADAD hNPCs (neuron/glia – GFP, green) and iMGLs (IBA1, red), stained for Aβ (blue). (K) Analysis of Aβ40 and Aβ42 expression in conditioned media of triculture model after 24 hours of pre-treatments followed by 3 hours of co-retreatments. Control IgG (n=5) and SEMA6D (n=5). Vertical bars; median. Unpaired two-tailed t-test.
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(A) Phagocytosis of synaptosomes by WT or TREM2 KO iMGL treated with SEMA6D (5 μg/ml). y-axis shows total well red fluorescent I.I., integrated intensity. Data averaged from three independent experiments (shown in fig. S11 A ); mean ± SD values. (B) Quantification of phagocytosis of synaptosomes by WT or TREM2 KO iMGL treated with SEMA6D at 24 hr. Data represent the three independent experiments performed in triplicate; median ± interquartile values at 24 hrs, p-values obtained from using a linear mixed-effects model (shown in fig. S11 A ). (C) Quantification of TNF-α and IL-6 in media from WT or TREM2 KO iMGL treated with SEMA6D (10 μg/ml), using the Mesoscale V-plex neuroinflammation panel. Boxplots indicate median and interquartile ranges, p-values calculated using linear regression. (D) Representative western blots of p-SYK and total SYK in WT and TREM2 KO iMGL treated with SEMA6D (10 μg/ml). β-actin as loading control (E) Quantification of p-SYK/total SYK chemiluminescent western blot analysis, n=3 (individual replicates shown in fig. S12 ). (F) Bulk RNA-seq effect size distribution indicating the transcriptional changes induced by SEMA6D treatment in WT and TREM2 KO iMGL. (G) Transcriptional changes in the TREM2 crosstalk sub-network associated with SEMA6D treatment (WT or TREM2 KO vs. untreated control) or TREM2 KO (TREM2 KO vs. WT, no treatment). Only TREM2 sub-network genes differentially expressed in at least one comparison were included. (H) Transcriptional effects of the same conditions across clinically and biologically relevant gene signatures. Background corresponds to 500 randomly selected genes. Solid dots correspond to a 1% FDR significance threshold for comparing the effect size distribution to the corresponding background (Mann-Whitney test). (I) Transcriptional effects of the experimental conditions across a representative subset of highly differentially expressed genes from the signatures in (H). (J) Representative confocal immunofluorescence of 3D triculture model consisting of differentiated ADAD hNPCs (neuron/glia – GFP, green) and iMGLs (IBA1, red), stained for Aβ (blue). (K) Analysis of Aβ40 and Aβ42 expression in conditioned media of triculture model after 24 hours of pre-treatments followed by 3 hours of co-retreatments. Control IgG (n=5) and SEMA6D (n=5). Vertical bars; median. Unpaired two-tailed t-test.

Journal: Science translational medicine

Article Title: Systematic analysis of cellular crosstalk reveals a role for SEMA6D-TREM2 regulating microglial function in Alzheimer’s disease

doi: 10.1126/scitranslmed.adx0027

Figure Lengend Snippet: (A) Phagocytosis of synaptosomes by WT or TREM2 KO iMGL treated with SEMA6D (5 μg/ml). y-axis shows total well red fluorescent I.I., integrated intensity. Data averaged from three independent experiments (shown in fig. S11 A ); mean ± SD values. (B) Quantification of phagocytosis of synaptosomes by WT or TREM2 KO iMGL treated with SEMA6D at 24 hr. Data represent the three independent experiments performed in triplicate; median ± interquartile values at 24 hrs, p-values obtained from using a linear mixed-effects model (shown in fig. S11 A ). (C) Quantification of TNF-α and IL-6 in media from WT or TREM2 KO iMGL treated with SEMA6D (10 μg/ml), using the Mesoscale V-plex neuroinflammation panel. Boxplots indicate median and interquartile ranges, p-values calculated using linear regression. (D) Representative western blots of p-SYK and total SYK in WT and TREM2 KO iMGL treated with SEMA6D (10 μg/ml). β-actin as loading control (E) Quantification of p-SYK/total SYK chemiluminescent western blot analysis, n=3 (individual replicates shown in fig. S12 ). (F) Bulk RNA-seq effect size distribution indicating the transcriptional changes induced by SEMA6D treatment in WT and TREM2 KO iMGL. (G) Transcriptional changes in the TREM2 crosstalk sub-network associated with SEMA6D treatment (WT or TREM2 KO vs. untreated control) or TREM2 KO (TREM2 KO vs. WT, no treatment). Only TREM2 sub-network genes differentially expressed in at least one comparison were included. (H) Transcriptional effects of the same conditions across clinically and biologically relevant gene signatures. Background corresponds to 500 randomly selected genes. Solid dots correspond to a 1% FDR significance threshold for comparing the effect size distribution to the corresponding background (Mann-Whitney test). (I) Transcriptional effects of the experimental conditions across a representative subset of highly differentially expressed genes from the signatures in (H). (J) Representative confocal immunofluorescence of 3D triculture model consisting of differentiated ADAD hNPCs (neuron/glia – GFP, green) and iMGLs (IBA1, red), stained for Aβ (blue). (K) Analysis of Aβ40 and Aβ42 expression in conditioned media of triculture model after 24 hours of pre-treatments followed by 3 hours of co-retreatments. Control IgG (n=5) and SEMA6D (n=5). Vertical bars; median. Unpaired two-tailed t-test.

Article Snippet: Novogene conducted iMGL cell sequencing and preliminary RNA-seq data processing.

Techniques: Activation Assay, Western Blot, Control, RNA Sequencing, Comparison, MANN-WHITNEY, Immunofluorescence, Staining, Expressing, Two Tailed Test