Identification and therapeutic modulation of a pro-inflammatory subset of disease-associated-microglia in Alzheimer's disease methods
Aim. Evidence-backed execution summary for Identification and therapeutic modulation of a pro-inflammatory subset of disease-associated-microglia in Alzheimer's disease methods from Identification and therapeutic modulation of a pro-inflammatory subset of disease-associated-microglia in Alzheimer's disease.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Methods
reagent used in the protocol.
- Use
- Weighted co-expression network analysis (WGCNA) was applied to existing microglial transcriptomic datasets from neuroinflammatory and neurodegenerative disease mouse models to identify modules of highly co-expressed genes. These modules were contrasted with known signatures of homeostatic microglia and DAM to reveal...
Flow cytometric and immunohistochemistry antibodies
reagent used in the protocol.
- Use
- Monoclonal fluorophore-conjugated antibodies used for flow cytometry were obtained from BD Biosciences (CD11b-APC-Cy7, CD45-PeCy7, CD11c-BV421, CD274-APC, CD69-APC, CD44-PeCy7 or FITC, CD184-PE, CD3e-FITC, Ly-6c-PE, Ly-6G-APC) and BioLegend (CD8a-BV785, CD45-PerCP, and ENPP1-BV421). For immunohistochemistry, rabbit...
Primary microglial isolation
reagent used in the protocol.
- Use
- Adult C57BL6 mice, age-matched female 5xFAD mice were euthanized and brains were isolated following rapid cold saline cardiac perfusion and CNS immune cells were isolated as previously described [, ]. Briefly, brains were minced over a 40 µm cell strainer and single cell suspensions were washed in PBS in...
Microglial Aβ42 phagocytosis assay
reagent used in the protocol.
- Use
- The ability of acutely isolated CNS mononuclear cells from wild type, LPS-treated and age-matched 5xFAD mice to phagocytose fluorescent-labeled Aβ fibrils ex-vivo was assessed by a flow cytometric assay. Fluorescent-labeled Aβ42 fibrils were prepared by co-incubating unlabeled and Hilyte488-labeled Aβ...
GEO datasets, microarray normalization, and batch correction
reagent used in the protocol.
- Use
- In a separate replication analysis, transcript count data from 64 purified microglial medium-throughput sequencing (Nanostring) studies (GEO dataset GSE101689 ) were obtained from supplemental data [ ] which were log2-transformed (0 counts set to 0.5) and batch-corrected using ComBat and assumption of common samples...
In-vitro assays of fAβ42 degradation and reactive oxygen species production by microglia
reagent used in the protocol.
- Use
- Bv2 microglia were maintained in Dulbecco's modified Eagle Medium (with 10% FBS). For in-vitro experiments, Bv2 cells were grown on glass cover slips, loaded with fluorescent Aβ42 fibrils and cultured up to 72 h. At different time points 0.5, 6, 16, 24, 48 and 72 h, the BV2 microglia were fixed...
Immunofluorescence microscopy
reagent used in the protocol.
- Use
- Double antigen immunofluorescence for Aβ and Iba1 in slides from ShK-223 and PBS treated mice involved antigen retrieval with 45% formic acid for 5 min followed by heating in microwave for 10 min in 10 mM citric acid (pH 6.0) solution. This was followed by similar blocking steps as outlined...
Flow cytometric studies
reagent used in the protocol.
- Use
- Freshly isolated CNS immune cells were labeled for CD11b, CD45, CD11c, CD44, CD184 (CXCR4), CD69 and CD274 using well characterized fluorophore-conjugated monoclonal antibodies. Kv1.3 channels expression on cell surface was assessed using fluorescein-conjugated ShK analog as stated above. Compensation experiments we...
Flow cytometric and immunohistochemistry antibodies
Monoclonal fluorophore-conjugated antibodies used for flow cytometry were obtained from BD Biosciences (CD11b-APC-Cy7, CD45-PeCy7, CD11c-BV421, CD274-APC, CD69-APC, CD44-PeCy7 or FITC, CD184-PE, CD3e-FITC, Ly-6c-PE, Ly-6G-APC) and BioLegend (CD8a-BV785, CD45-PerCP, and ENPP1-BV421). For immunohistochemistry, rabbit...
- Use
- Monoclonal fluorophore-conjugated antibodies used for flow cytometry were obtained from BD Biosciences (CD11b-APC-Cy7, CD45-PeCy7, CD11c-BV421, CD274-APC, CD69-APC, CD44-PeCy7 or FITC, CD184-PE, CD3e-FITC, Ly-6c-PE, Ly-6G-APC) and BioLegend (CD8a-BV785, CD45-PerCP, and ENPP1-BV421). For immunohistochemistry, rabbit...
Primary microglial isolation
Adult C57BL6 mice, age-matched female 5xFAD mice were euthanized and brains were isolated following rapid cold saline cardiac perfusion and CNS immune cells were isolated as previously described [, ]. Briefly, brains were minced over a 40 µm cell strainer and single cell suspensions were washed in PBS in...
- Use
- Adult C57BL6 mice, age-matched female 5xFAD mice were euthanized and brains were isolated following rapid cold saline cardiac perfusion and CNS immune cells were isolated as previously described [, ]. Briefly, brains were minced over a 40 µm cell strainer and single cell suspensions were washed in PBS in...
Microglial Aβ42 phagocytosis assay
The ability of acutely isolated CNS mononuclear cells from wild type, LPS-treated and age-matched 5xFAD mice to phagocytose fluorescent-labeled Aβ fibrils ex-vivo was assessed by a flow cytometric assay. Fluorescent-labeled Aβ42 fibrils were prepared by co-incubating unlabeled and Hilyte488-labeled Aβ...
- Use
- The ability of acutely isolated CNS mononuclear cells from wild type, LPS-treated and age-matched 5xFAD mice to phagocytose fluorescent-labeled Aβ fibrils ex-vivo was assessed by a flow cytometric assay. Fluorescent-labeled Aβ42 fibrils were prepared by co-incubating unlabeled and Hilyte488-labeled Aβ...
GEO datasets, microarray normalization, and batch correction
Microarray transcriptomic datasets were obtained from the genomics data repository Gene Expression Omnibus (GEO). Microarray transcriptome datasets of FACS-purified microglia from 8.5 mo old WT ( n = 5), TREM2 -/- ( n = 4), 5xFAD (n = 5) and TREM2 -/- /5x...
- Use
- Microarray transcriptomic datasets were obtained from the genomics data repository Gene Expression Omnibus (GEO). Microarray transcriptome datasets of FACS-purified microglia from 8.5 mo old WT ( n = 5), TREM2 -/- ( n = 4), 5xFAD (n = 5) and TREM2 -/- /5x...
GEO datasets, microarray normalization, and batch correction
Agilent microarray data from microglia isolated from WT and APP/PS1 mice ( n = 7 each) were obtained as a subset of the samples in GEO dataset GSE74615. Agilent green (Cy3) and red (Cy5) channels were background corrected according to the normexp method in limma::backgroundCorrect() function, [ ] then...
- Use
- Agilent microarray data from microglia isolated from WT and APP/PS1 mice ( n = 7 each) were obtained as a subset of the samples in GEO dataset GSE74615. Agilent green (Cy3) and red (Cy5) channels were background corrected according to the normexp method in limma::backgroundCorrect() function, [ ] then...
GEO datasets, microarray normalization, and batch correction
In a separate replication analysis, transcript count data from 64 purified microglial medium-throughput sequencing (Nanostring) studies (GEO dataset GSE101689 ) were obtained from supplemental data [ ] which were log2-transformed (0 counts set to 0.5) and batch-corrected using ComBat and assumption of common samples...
- Use
- In a separate replication analysis, transcript count data from 64 purified microglial medium-throughput sequencing (Nanostring) studies (GEO dataset GSE101689 ) were obtained from supplemental data [ ] which were log2-transformed (0 counts set to 0.5) and batch-corrected using ComBat and assumption of common samples...
WGCNA
The R package WGCNA was used to construct a co-expression network using normalized and batch-corrected data of the 43 microarray-measured samples just described using a previously published approach [ ]. A threshold power of 10 was chosen since it was the smallest threshold that resulted in a scale-free R 2 fit of 0...
- Use
- The R package WGCNA was used to construct a co-expression network using normalized and batch-corrected data of the 43 microarray-measured samples just described using a previously published approach [ ]. A threshold power of 10 was chosen since it was the smallest threshold that resulted in a scale-free R 2 fit of 0...
In-vitro assays of fAβ42 degradation and reactive oxygen species production by microglia
Bv2 microglia were maintained in Dulbecco's modified Eagle Medium (with 10% FBS). For in-vitro experiments, Bv2 cells were grown on glass cover slips, loaded with fluorescent Aβ42 fibrils and cultured up to 72 h. At different time points 0.5, 6, 16, 24, 48 and 72 h, the BV2 microglia were fixed...
- Use
- Bv2 microglia were maintained in Dulbecco's modified Eagle Medium (with 10% FBS). For in-vitro experiments, Bv2 cells were grown on glass cover slips, loaded with fluorescent Aβ42 fibrils and cultured up to 72 h. At different time points 0.5, 6, 16, 24, 48 and 72 h, the BV2 microglia were fixed...
Other statistical considerations
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Graphpad Prism (Ver. 5), Cytoscape (Ver. 3.5.1) and SPSS (Ver. 22) were used to create graphs and perform statistical analyses. All data are shown as mean ± SEM. For experiments with > 2 groups, one way ANOVA was performed to detect differences across groups and post-hoc pairwise comparisons w...
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Primary microglial isolation
Adult C57BL6 mice, age-matched female 5xFAD mice were euthanized and brains were isolated following rapid cold saline cardiac perfusion and CNS immune cells were isolated as previously described [, ]. Briefly, brains were minced over a 40 µm cell strainer and single cell suspensions were washed in PBS in a centrifuge for 5 min at 800 × g at room temperature. Supernatants were discarded and cell pellets re-suspended in 6 mL of 37% stock isotonic Percoll (SIP) solution (90% Percoll + 10% 10X HBSS) per brain. The cell suspension was transferred into 15 mL conical tubes and 2 mL of 70% SIP slowly under laid. Then on top of the 37% layer, 2 mL of 30% SIP was slowly layered. The established gradient was then centrifuged for 25 min at 800 × g with zero deceleration at 20 °C. Floating myelin in the top layer was then removed and a...
Microglial Aβ42 phagocytosis assay
The ability of acutely isolated CNS mononuclear cells from wild type, LPS-treated and age-matched 5xFAD mice to phagocytose fluorescent-labeled Aβ fibrils ex-vivo was assessed by a flow cytometric assay. Fluorescent-labeled Aβ42 fibrils were prepared by co-incubating unlabeled and Hilyte488-labeled Aβ42 required unlabeled Aβ42 (Anaspec Cat # AS-60479-01) at a 1:4 ratio. Purified Aβ42 was first linearized in 10% w / v NH 4 OH and re-lyophilized to reduce any preexisting aggregates. 0.5 mg of unlabeled lyophilized Aβ42 was then reconstituted to 150 µM by adding 1 mM NaOH, bath sonicated for 10 min followed by addition of 10X buffer (100 mM sodium phosphate, pH 7.1) to bring the final pH to 7.4. 410.64 µL of that aliquot was added to 0.1 mg of HiLyte-488 Aβ42 to achieve a total of 200 µM Aβ...
Animals
Female C57BL/6 J and female 5xFAD mice used for the studies were housed in the Department of Animal Resources at Emory University under standard conditions with no special food/water accommodations. Institutional Animal Care and Use Committee approval was obtained prior to in-vivo work and all work was performed in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Adult mice were given intraperitoneal LPS injections (10 µg/dose × 4 daily doses) to induce acute neuroinflammation [, ]. If ≥25% of weight loss was observed, animals were euthanized. In some experiments, 5xFAD mice received i.p. doses of T0901317 (30 mg/kg) twice a week for 2 weeks.
WGCNA
The R package WGCNA was used to construct a co-expression network using normalized and batch-corrected data of the 43 microarray-measured samples just described using a previously published approach [ ]. A threshold power of 10 was chosen since it was the smallest threshold that resulted in a scale-free R 2 fit of 0.8. The network was created using WGCNA::blockwiseModules() function, in a single block (maxBlockSize > 16,721). Briefly, this function calculated topologic overlap (TO) with bicor correlation function, then genes were hierarchically clustered using 1-TO (dissTOM) as the distance measure. Initial module assignments were determined by using dynamic tree-cutting, using default parameters except deepSplit = 2, mergeCutHeight =0.20, minModulesize = 40, pamStage = TRUE, and pamRespectsDendro = TRUE), but genes were allowed to...
Long-term ShK-223 treatment trial
Adult female 5xFAD mice aged 3 mo were treated intraperitoneally twice a week until 6 mo of age with PBS or ShK-223 (dose 100 µg/kg), n = 10 each and also age-matched 5 female C57BL/6 mice were included in the study. At 6 mo, neurobehavioral testing including Fear conditioning and Morris Water Maze was performed. The mice were euthanized, brains isolated and the brains were utilized for immunohistochemistry studies.
Reverse transcriptase quantitative PCR
CNS immune cells isolated from age-matched C57BL6 mice treated with four once daily intraperitoneal doses of sterile PBS, ShK-223 (100 µg/kg), LPS (20 µg/dose) or co-administered LPS + ShK-223 ( n = 3/group) were washed in PBS containing RNAase inhibitors and used for RT-qPCR using our published protocols [ ]. For total RNA extraction from cells, 1 mL Trizol was added to the cells, the pellets were homogenized in the Trizol by repetitive pipetting and incubated for at least 1 h at room temperature. Then, 0.2 mL of chloroform per 1 mL of Trizol was added and incubated for 2-3 min followed by centrifugation at 12000 x g for 15 min at 4 °C to obtain an upper colorless aqueous phase, an interphase and a lower red phenol chloroform phase. RNA in the aqueous phase was transferred to other tubes. 0.5&#...
Validation of Kv1.3 channels as specific regulators of pro-inflammatory DAM responses and neuropathology in AD mouse...
Next, we performed in-vivo studies to determine whether Kv1.3 channel inhibition by ShK-223 can inhibit pro-inflammatory DAM and augment anti-inflammatory DAM gene expression in the model of acute neuroinflammation induced by low dose systemic LPS administration [, ]. We found that ShK-223 treatment inhibited LPS-induced upregulation of proinflammatory genes (Ptgs2, Il1b) and augmented expression of anti-inflammatory DAM genes (Kcnj2, Nceh1, Timp2) without affecting LPS-induced suppression of homeostatic gene Tmem119 (Fig. ). The ability of systemic ShK-223 to modulate microglial function in this model indirectly supports its CNS bioavailability.
Measurement outputs
What raw and processed outputs should exist?
Weighted co-expression network analysis (WGCNA) was applied to existing microglial transcriptomic datasets from neuroinflammatory and neurodegenerative disease mouse models to i...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Microarray transcriptomic datasets were obtained from the genomics data repository Gene Expression Omnibus (GEO). Microarray transcriptome datasets of FACS-purified microglia fr...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Agilent microarray data from microglia isolated from WT and APP/PS1 mice ( n = 7 each) were obtained as a subset of the samples in GEO dataset GSE74615. Agilent gr...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
In a separate replication analysis, transcript count data from 64 purified microglial medium-throughput sequencing (Nanostring) studies (GEO dataset GSE101689 ) were obtained fr...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
The patient and pathological characteristics of the 47 post-mortem BLSA samples used for proteomics and the methods for tandem-mass-tag quantitative proteomics, subsequent analyses and WGCNA are available online ( https://www.synapse.org/#!Synapse:syn11209141 ) and will be pub...
from paperScoring or quantification
Quantify the primary readouts for this experiment: Weighted co-expression network analysis (WGCNA) was applied to existing microglial transcriptomic datasets from neuroinflammatory and neurodegenerative disease mouse models to i...; Microarray transcriptomic datasets were obtained from the genomics data repository Gene Expression Omnibus (GEO). Microarray transcriptome datasets of FACS-purified microglia fr...; Agilent microarray data from microglia isolated from WT and APP/PS1 mice ( n = 7 each) were obtained as a subset of the samples in GEO dataset GSE74615. Agilent gr...; In a separate replication analysis, transcript count data from 64 purified microglial medium-throughput sequencing (Nanostring) studies (GEO dataset GSE101689 ) were obtained fr....
from paperStatistical comparison
The patient and pathological characteristics of the 47 post-mortem BLSA samples used for proteomics and the methods for tandem-mass-tag quantitative proteomics, subsequent analy...; Graphpad Prism (Ver. 5), Cytoscape (Ver. 3.5.1) and SPSS (Ver. 22) were used to create graphs and perform statistical analyses. All data are shown as mean ± SEM...; To identify therapeutic approaches to specifically inhibit pro-inflammatory DAM and promote anti-inflammatory DAM gene expression, we performed an analysis using the connectivit...; To confirm the presence of distinct pro-inflammatory and anti-inflammatory DAM subsets resolved by co-expression analyses, we performed flow cytometric studies of acutely isolat...
from paperReporting output
Report representative outputs alongside summary comparisons for Weighted co-expression network analysis (WGCNA) was applied to existing microglial transcriptomic datasets from neuroinflammatory and neurodegenerative disease mouse models to i..., Microarray transcriptomic datasets were obtained from the genomics data repository Gene Expression Omnibus (GEO). Microarray transcriptome datasets of FACS-purified microglia fr..., Agilent microarray data from microglia isolated from WT and APP/PS1 mice ( n = 7 each) were obtained as a subset of the samples in GEO dataset GSE74615. Agilent gr..., In a separate replication analysis, transcript count data from 64 purified microglial medium-throughput sequencing (Nanostring) studies (GEO dataset GSE101689 ) were obtained fr....
inferred from protocolStructured statistical methods
The patient and pathological characteristics of the 47 post-mortem BLSA samples used for proteomics and the methods for tandem-mass-tag quantitative proteomics, subsequent analy...; Graphpad Prism (Ver. 5), Cytoscape (Ver. 3.5.1) and SPSS (Ver. 22) were used to create graphs and perform statistical analyses. All data are shown as mean ± SEM...; To identify therapeutic approaches to specifically inhibit pro-inflammatory DAM and promote anti-inflammatory DAM gene expression, we performed an analysis using the connectivit...; To confirm the presence of distinct pro-inflammatory and anti-inflammatory DAM subsets resolved by co-expression analyses, we performed flow cytometric studies of acutely isolat...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (7)
Adult C57BL6 mice, age-matched female 5xFAD mice were euthanized and brains were isolated following rapid cold saline cardiac perfusion and CNS immune cells were isolated as previously described [, ]. Briefly, brains were minced over a 40 µm cell strainer and single cell suspensions were washed in PBS in a centrifuge for 5 min at 800 × g at room temperature. Supernatants were discarded and cell pellets re-suspended in 6 mL of 37% stock isotonic Percoll (SIP) solution (90% Percoll + 10% 10X HBSS) per brain. The cell suspension was transferred into 15 mL conical tubes and 2 mL of 70% SIP slowly under laid. Then on top of the 37% layer, 2 mL of 30% SIP was slowly layered. The established gradient was then centrifuged for 25 min at 800 × g with zero deceleration at 20 °C. Floating myelin in the top layer was then removed and a Pasteur pipette used to carefully collect 3 mL from the 70-37% interphase without disturbing the 70% layer. Cells were then washed × 3 in 10 mL cold PBS and the pellets comprising of CNS immune cells were then re-suspended in 100 µL of the appropriate buffer.
The ability of acutely isolated CNS mononuclear cells from wild type, LPS-treated and age-matched 5xFAD mice to phagocytose fluorescent-labeled Aβ fibrils ex-vivo was assessed by a flow cytometric assay. Fluorescent-labeled Aβ42 fibrils were prepared by co-incubating unlabeled and Hilyte488-labeled Aβ42 required unlabeled Aβ42 (Anaspec Cat # AS-60479-01) at a 1:4 ratio. Purified Aβ42 was first linearized in 10% w / v NH 4 OH and re-lyophilized to reduce any preexisting aggregates. 0.5 mg of unlabeled lyophilized Aβ42 was then reconstituted to 150 µM by adding 1 mM NaOH, bath sonicated for 10 min followed by addition of 10X buffer (100 mM sodium phosphate, pH 7.1) to bring the final pH to 7.4. 410.64 µL of that aliquot was added to 0.1 mg of HiLyte-488 Aβ42 to achieve a total of 200 µM Aβ42 solution. The sample was incubated in dark at room temperature for 5-7 days before being transferred to 4 °C. Phagocytosis of fluorescent-labeled Aβ42 fibrils (fAβ42-HiLyte488) was performed by incubating acutely isolated CNS immune cells (pretreated ex-vivo with S...
Female C57BL/6 J and female 5xFAD mice used for the studies were housed in the Department of Animal Resources at Emory University under standard conditions with no special food/water accommodations. Institutional Animal Care and Use Committee approval was obtained prior to in-vivo work and all work was performed in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Adult mice were given intraperitoneal LPS injections (10 µg/dose × 4 daily doses) to induce acute neuroinflammation [, ]. If ≥25% of weight loss was observed, animals were euthanized. In some experiments, 5xFAD mice received i.p. doses of T0901317 (30 mg/kg) twice a week for 2 weeks.
The R package WGCNA was used to construct a co-expression network using normalized and batch-corrected data of the 43 microarray-measured samples just described using a previously published approach [ ]. A threshold power of 10 was chosen since it was the smallest threshold that resulted in a scale-free R 2 fit of 0.8. The network was created using WGCNA::blockwiseModules() function, in a single block (maxBlockSize > 16,721). Briefly, this function calculated topologic overlap (TO) with bicor correlation function, then genes were hierarchically clustered using 1-TO (dissTOM) as the distance measure. Initial module assignments were determined by using dynamic tree-cutting, using default parameters except deepSplit = 2, mergeCutHeight =0.20, minModulesize = 40, pamStage = TRUE, and pamRespectsDendro = TRUE), but genes were allowed to be reassigned to modules with better correlation if p < 0.05 for those correlations. The network type was signed, so that anticorrelated genes were not assigned to the same module. Resulting 19 modules or groups of co-expressed genes ranging in size from 2165 genes (turquoise) to 75 ge...
Adult female 5xFAD mice aged 3 mo were treated intraperitoneally twice a week until 6 mo of age with PBS or ShK-223 (dose 100 µg/kg), n = 10 each and also age-matched 5 female C57BL/6 mice were included in the study. At 6 mo, neurobehavioral testing including Fear conditioning and Morris Water Maze was performed. The mice were euthanized, brains isolated and the brains were utilized for immunohistochemistry studies.
CNS immune cells isolated from age-matched C57BL6 mice treated with four once daily intraperitoneal doses of sterile PBS, ShK-223 (100 µg/kg), LPS (20 µg/dose) or co-administered LPS + ShK-223 ( n = 3/group) were washed in PBS containing RNAase inhibitors and used for RT-qPCR using our published protocols [ ]. For total RNA extraction from cells, 1 mL Trizol was added to the cells, the pellets were homogenized in the Trizol by repetitive pipetting and incubated for at least 1 h at room temperature. Then, 0.2 mL of chloroform per 1 mL of Trizol was added and incubated for 2-3 min followed by centrifugation at 12000 x g for 15 min at 4 °C to obtain an upper colorless aqueous phase, an interphase and a lower red phenol chloroform phase. RNA in the aqueous phase was transferred to other tubes. 0.5 mL of 100% iso-propranolol was added to the aqueous phase per 1 mL of Trizol used and the mixture was incubated for 10 min and centrifuged at 12000 x g for 10 min at 4 °C. The pellet was washed with 1 mL of 75% ethanol per 1 mL of Trizol used (initial homogeniz...
Next, we performed in-vivo studies to determine whether Kv1.3 channel inhibition by ShK-223 can inhibit pro-inflammatory DAM and augment anti-inflammatory DAM gene expression in the model of acute neuroinflammation induced by low dose systemic LPS administration [, ]. We found that ShK-223 treatment inhibited LPS-induced upregulation of proinflammatory genes (Ptgs2, Il1b) and augmented expression of anti-inflammatory DAM genes (Kcnj2, Nceh1, Timp2) without affecting LPS-induced suppression of homeostatic gene Tmem119 (Fig. ). The ability of systemic ShK-223 to modulate microglial function in this model indirectly supports its CNS bioavailability.
Machine-readable layer
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"@type": "HowTo",
"name": "Identification and therapeutic modulation of a pro-inflammatory subset of disease-associated-microglia in Alzheimer's disease methods",
"description": "Evidence-backed execution summary for Identification and therapeutic modulation of a pro-inflammatory subset of disease-associated-microglia in Alzheimer's disease methods from Identification and therapeutic modulation of a pro-inflammatory subset of disease-associated-microglia in Alzheimer's disease.",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Primary microglial isolation",
"text": "Adult C57BL6 mice, age-matched female 5xFAD mice were euthanized and brains were isolated following rapid cold saline cardiac perfusion and CNS immune cells were isolated as previously described [, ]. Briefly, brains were minced over a 40 µm cell strainer and single cell suspensions were washed in PBS in a centrifuge for 5 min at 800 × g at room temperature. Supernatants were discarded and cell pellets re-suspended in 6 mL of 37% stock isotonic Percoll (SIP) solution (90% Percoll + 10% 10X HBSS) per brain. The cell suspension was transferred into 15 mL conical tubes and 2 mL of 70% SIP slowly under laid. Then on top of the 37% layer, 2 mL of 30% SIP was slowly layered. The established gradient was then centrifuged for 25 min at 800 × g with zero deceleration at 20 °C. Floating myelin in the top layer was then removed and a..."
},
{
"@type": "HowToStep",
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"name": "Microglial Aβ42 phagocytosis assay",
"text": "The ability of acutely isolated CNS mononuclear cells from wild type, LPS-treated and age-matched 5xFAD mice to phagocytose fluorescent-labeled Aβ fibrils ex-vivo was assessed by a flow cytometric assay. Fluorescent-labeled Aβ42 fibrils were prepared by co-incubating unlabeled and Hilyte488-labeled Aβ42 required unlabeled Aβ42 (Anaspec Cat # AS-60479-01) at a 1:4 ratio. Purified Aβ42 was first linearized in 10% w / v NH 4 OH and re-lyophilized to reduce any preexisting aggregates. 0.5 mg of unlabeled lyophilized Aβ42 was then reconstituted to 150 µM by adding 1 mM NaOH, bath sonicated for 10 min followed by addition of 10X buffer (100 mM sodium phosphate, pH 7.1) to bring the final pH to 7.4. 410.64 µL of that aliquot was added to 0.1 mg of HiLyte-488 Aβ42 to achieve a total of 200 µM Aβ..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Animals",
"text": "Female C57BL/6 J and female 5xFAD mice used for the studies were housed in the Department of Animal Resources at Emory University under standard conditions with no special food/water accommodations. Institutional Animal Care and Use Committee approval was obtained prior to in-vivo work and all work was performed in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Adult mice were given intraperitoneal LPS injections (10 µg/dose × 4 daily doses) to induce acute neuroinflammation [, ]. If ≥25% of weight loss was observed, animals were euthanized. In some experiments, 5xFAD mice received i.p. doses of T0901317 (30 mg/kg) twice a week for 2 weeks."
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"name": "WGCNA",
"text": "The R package WGCNA was used to construct a co-expression network using normalized and batch-corrected data of the 43 microarray-measured samples just described using a previously published approach [ ]. A threshold power of 10 was chosen since it was the smallest threshold that resulted in a scale-free R 2 fit of 0.8. The network was created using WGCNA::blockwiseModules() function, in a single block (maxBlockSize > 16,721). Briefly, this function calculated topologic overlap (TO) with bicor correlation function, then genes were hierarchically clustered using 1-TO (dissTOM) as the distance measure. Initial module assignments were determined by using dynamic tree-cutting, using default parameters except deepSplit = 2, mergeCutHeight =0.20, minModulesize = 40, pamStage = TRUE, and pamRespectsDendro = TRUE), but genes were allowed to..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Long-term ShK-223 treatment trial",
"text": "Adult female 5xFAD mice aged 3 mo were treated intraperitoneally twice a week until 6 mo of age with PBS or ShK-223 (dose 100 µg/kg), n = 10 each and also age-matched 5 female C57BL/6 mice were included in the study. At 6 mo, neurobehavioral testing including Fear conditioning and Morris Water Maze was performed. The mice were euthanized, brains isolated and the brains were utilized for immunohistochemistry studies."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Reverse transcriptase quantitative PCR",
"text": "CNS immune cells isolated from age-matched C57BL6 mice treated with four once daily intraperitoneal doses of sterile PBS, ShK-223 (100 µg/kg), LPS (20 µg/dose) or co-administered LPS + ShK-223 ( n = 3/group) were washed in PBS containing RNAase inhibitors and used for RT-qPCR using our published protocols [ ]. For total RNA extraction from cells, 1 mL Trizol was added to the cells, the pellets were homogenized in the Trizol by repetitive pipetting and incubated for at least 1 h at room temperature. Then, 0.2 mL of chloroform per 1 mL of Trizol was added and incubated for 2-3 min followed by centrifugation at 12000 x g for 15 min at 4 °C to obtain an upper colorless aqueous phase, an interphase and a lower red phenol chloroform phase. RNA in the aqueous phase was transferred to other tubes. 0.5&#..."
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{
"@type": "HowToStep",
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"text": "Next, we performed in-vivo studies to determine whether Kv1.3 channel inhibition by ShK-223 can inhibit pro-inflammatory DAM and augment anti-inflammatory DAM gene expression in the model of acute neuroinflammation induced by low dose systemic LPS administration [, ]. We found that ShK-223 treatment inhibited LPS-induced upregulation of proinflammatory genes (Ptgs2, Il1b) and augmented expression of anti-inflammatory DAM genes (Kcnj2, Nceh1, Timp2) without affecting LPS-induced suppression of homeostatic gene Tmem119 (Fig. ). The ability of systemic ShK-223 to modulate microglial function in this model indirectly supports its CNS bioavailability."
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