e coli lipopolysaccharide  (InvivoGen)

Journal: bioRxiv

Article Title: New Human IPSC Models of Late-onset Alzheimer’s Disease Polygenic Risk Identify Multiple Impairments of Microglial Function

doi: 10.64898/2026.04.29.721607

Figure Lengend Snippet: ( A ) Total ATP production rate measured in the Seahorse XF Real-Time ATP Rate assay, with and without LPS stimulation (20ng/mL for 18 hours). Table shows results from the linear mixed effects model, including corrected and uncorrected p values. ( B ) ATP production rate from glycolysis (glycoATP) and ( C ) ATP production rate from mitochondrial respiration (mitoATP) measured in the Seahorse XF Real-Time ATP Rate assay, with and without LPS challenge (20ng/mL for 18 hours). ( D ) Fold change of the LPS-challenged ATP Rate assay data relative to unstimulated (raw data presented in ( A-C )). For ( A-D ), data was first normalised to cell count, then a median obtained from 3-10 replicate wells for each experiments. ( E ) Total mitochondrial content (measured by mean fluorescence intensity of MitoTracker Green) and ( F ) mitochondrial network width:length ratio (measured using Mitotracker Green morphology) with representative images (± FCCP) shown in (G) . (H) Mitochondrial membrane potential analysed by JC1 red/green fluorescence intensity, with representative images (± FCCP) shown in (I) . For (E, F and H) , data plotted is mean per cell, then medians obtained from 4 replicate wells for each experiment. Across all parts of the figure, box plots show the median and interquartile range for N = 35 HRLOAD, 17 LRWELL from a minimum of 4 experiments. HRLOAD (red) was compared to LRWELL (blue), and LPS-challenged to unstimulated, using linear mixed effects modelling with p-values corrected by Benjamini-Hochberg procedure. * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bars: 20 µm.

Article Snippet: On day 9, a full media change was performed with 150 μL of full microglia media +/- E. coli lipopolysaccharide (20 ng/mL, Invivogen, tlrl-eklps).

Techniques: Cell Characterization, Fluorescence, Membrane

Journal: bioRxiv

Article Title: New Human IPSC Models of Late-onset Alzheimer’s Disease Polygenic Risk Identify Multiple Impairments of Microglial Function

doi: 10.64898/2026.04.29.721607

Figure Lengend Snippet: (A) Representative ECAR and OCR trace for LPS stimulated LRWELL (DRICUi020-A) and HRLOAD (DRICUi006-A) iPSC-microglia in the Seahorse XF Real-Time ATP Rate assay. Data points show mean ± SEM of n=5-6 technical replicate wells from 1 assay. (B) Total, (C) mitochondrial (mitoATP), and (D) glycolytic (glycoATP) ATP production rates (pmol/min) were measured using a Seahorse XF Real-Time ATP Rate assay, with LPS stimulation, showing that APOE ε4 does not alter the HRLOAD phenotype. Data plotted is medians obtained from 3-10 replicate wells. Across all parts of the figure, box plots show the median and interquartile range for N= 17 HRLOAD APOE ε33, 17 HRLOAD APOE ε34/44, 17 LRWELL from a minimum of 4 experiments. LRWELL (blue), HRLOAD33 (orange), and HRLOAD34/44 (red) were compared using linear mixed effects modelling, with APOE as a fixed effect. Uncorrected p-values are displayed. * p < 0.05, ** p < 0.01.

Article Snippet: On day 9, a full media change was performed with 150 μL of full microglia media +/- E. coli lipopolysaccharide (20 ng/mL, Invivogen, tlrl-eklps).

Techniques:

Journal: bioRxiv

Article Title: New Human IPSC Models of Late-onset Alzheimer’s Disease Polygenic Risk Identify Multiple Impairments of Microglial Function

doi: 10.64898/2026.04.29.721607

Figure Lengend Snippet: Levels of secreted proteins were measured using a LegendPLEX cytometric bead array assay in unstimulated and LPS-challenged (20 ng/mL for 24 hours) iPSC microglia for ( A ) IL-6, (C) TNF, (E) sTREM2, ( F) IL-10, (G) CCL2, (H) IL-1β. HRLOAD (red) was compared to LRWELL (blue), and LPS-challenged to unstimulated, using linear mixed effects modelling with p-values corrected by Benjamini-Hochberg procedure. Exploratory analysis of LPS-challenged data looking at the effect of APOE ε4 is shown for significant HRLOAD:LPS interactions in IL-6 ( B ) and TNF ( D ). LRWELL (blue), HRLOAD33 (orange), and HRLOAD34/44 (red) were compared using linear mixed effects modelling, with APOE as a fixed effect. Uncorrected p-values are displayed. Across all parts of the figure, data plotted is the mean of 4 replicate wells that were pooled, then normalized to the average cell count across those wells. Box plots display the median and interquartile range of secreted protein per cell (pg/mL/cell) for N = 28 HRLOAD, 15 LRWELL from a minimum of 4 experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: On day 9, a full media change was performed with 150 μL of full microglia media +/- E. coli lipopolysaccharide (20 ng/mL, Invivogen, tlrl-eklps).

Techniques: Cell Characterization

Journal: bioRxiv

Article Title: New Human IPSC Models of Late-onset Alzheimer’s Disease Polygenic Risk Identify Multiple Impairments of Microglial Function

doi: 10.64898/2026.04.29.721607

Figure Lengend Snippet: Endocytosis was investigated by performing uptake assays of (A) HiLyte-488 Amyloid-β 1-42 peptide and (C) pHrodo Red-transferrin, measuring relative spot intensity. Representative images (± Dynasore to inhibit endocytosis) are shown in (B) and (D) for each assay, respectively. Phagocytosis was investigated by performing uptake assays of pHrodo Red-labelled (E) dead neuronal cells, (G) mouse myelin, and (I) E. coli bioparticles, measuring total spot area per cell. Representative images (± Cytochalasin D to inhibit phagocytosis) are shown in (F) , (H) and (J) for each assay, respectively. Across all parts of the figure, a median of 4 technical well replicates was first taken in each experiment. Box plots display the median and interquartile range for N= 35 HRLOAD, 17 LRWELL from a minimum of 4 experiments. For all phenotypes HRLOAD was compared to LRWELL using linear mixed effects modelling with p-values corrected by Benjamini-Hochberg procedure. *** p < 0.001. Scale bars: 20 µm.

Article Snippet: On day 9, a full media change was performed with 150 μL of full microglia media +/- E. coli lipopolysaccharide (20 ng/mL, Invivogen, tlrl-eklps).

Techniques:

Journal: bioRxiv

Article Title: New Human IPSC Models of Late-onset Alzheimer’s Disease Polygenic Risk Identify Multiple Impairments of Microglial Function

doi: 10.64898/2026.04.29.721607

Figure Lengend Snippet: (A) Correlation matrix for the significant or trending HRLOAD phenotypes in this paper, which are LPS-challenged mitochondrial ATP production rate (mitoATP), LPS-challenged glycolytic ATP production rate (glycoATP), unstimulated IBA1 expression, LPS-challenged IL-6 secretion, LPS-challenged TNF secretion, amyloid-β (Aβ) uptake, transferrin uptake. P values include a Bonferroni correction for multiple testing. Individual correlation plots are shown for (B) mitoATP production versus glycoATP production, (C) mitoATP production versus IBA1 median fluorescence intensity, (D) IL-6 versus TNF secretion, (E) Aβ versus transferrin uptake. For ( B-E ), data points represent a median of n=3-10 technical well replicates from one iPSC line in an individual matched experiment.

Article Snippet: On day 9, a full media change was performed with 150 μL of full microglia media +/- E. coli lipopolysaccharide (20 ng/mL, Invivogen, tlrl-eklps).

Techniques: Expressing, Fluorescence

Journal: bioRxiv

Article Title: New Human IPSC Models of Late-onset Alzheimer’s Disease Polygenic Risk Identify Multiple Impairments of Microglial Function

doi: 10.64898/2026.04.29.721607

Figure Lengend Snippet: (A) Principal component analysis shows clear separation by stimulation condition but not by genotype. (B) Volcano plot of differentially expressed proteins comparing stimulated HRLOAD to LRWELL, with the significant protein highlighted in red. (C,D) Pathway Enrichment of the Proteome. Bar charts show the most statistically significant Reactome terms filtered by a Jaccard similarity coefficient (cutoff < 0.4) to eliminate redundant gene sets and highlight distinct biological processes for (C) Unstimulated and (D) LPS-stimulated (20 ng/mL for 24 hours) samples. Proteins were ranked for GSEA using a composite score of p-value and Log Fold-Change, * indicates significant enrichment, other terms are not statistically significant. N= 30 HRLOAD, 16 LRWELL. Data is from 2-3 technical replicate wells processed separately from one harvest.

Article Snippet: On day 9, a full media change was performed with 150 μL of full microglia media +/- E. coli lipopolysaccharide (20 ng/mL, Invivogen, tlrl-eklps).

Techniques:

Journal: bioRxiv

Article Title: New Human IPSC Models of Late-onset Alzheimer’s Disease Polygenic Risk Identify Multiple Impairments of Microglial Function

doi: 10.64898/2026.04.29.721607

Figure Lengend Snippet: (A) Volcano plot of differentially expressed proteins comparing LPS-stimulated to unstimulated, significant upregulated and downregulated proteins are highlighted in red and blue respectively. For the phenotypic assays: (B) stimulated Mitochondrial ATP production (MitoATP), (C) stimulated IL-6 secretion, (D) Amyloid-β (AB) uptake, the top 5 highest and bottom 5 lowest-performing extreme lines were selected and proteins were ranked for GSEA using a composite score of p-value and Log Fold-Change. Bar charts show the most statistically significant Reactome terms filtered by a Jaccard similarity coefficient (cutoff < 0.4) to eliminate redundant gene sets and highlight distinct biological processes. Data is from 2-3 technical replicate wells processed separately from one harvest.

Article Snippet: On day 9, a full media change was performed with 150 μL of full microglia media +/- E. coli lipopolysaccharide (20 ng/mL, Invivogen, tlrl-eklps).

Techniques: