clu elisa kit  (R&D Systems)

Journal: medRxiv

Article Title: Alzheimer’s disease protective allele of Clusterin modulates neuronal excitability through lipid-droplet-mediated neuron-glia communication

doi: 10.1101/2024.08.14.24312009

Figure Lengend Snippet: (A) Schematic research design. ATAC-seq was performed to identify OCRs and GWAS risk SNPs that showed ASoC in iPSC Glutaminergic (iGlut), GABAergic (iGABA), and dopaminergic (iDA) neurons, microglia (iMG) and astrocytes (iAst), which is followed by transcription factor (TF) binding prediction to confirm putative functional AD risk SNP, CRIPSR-cas9 SNP editing, and functional assays in cells. (B) ASoC mapping identifies intronic rs1532278 as a putatively functional SNP among several other GWAS risk SNPs equally associated with AD at the CLU locus. Note that rs1532278 is the only SNP within the neuronal OCR with a stronger OCR peak in iGlut. (C) T and C alleles of rs1532278 show differential allelic ATAC-seq reads (i.e., ASoC) in iGlut. The bottom panel shows the two most conserved TF binding motifs at the SNP site. (D) Schematic CLU gene structure near rs1532278 (upper panel) and a diagram showing CRISPR-Cas9 editing of rs1532278 in two iPSC lines (CD05 and CD07; T/C) to covert T/C lines to isogenic T/T and C/C lines (middle panel). Representative images of iGlut of all three genotypes are also shown (bottom panel); MAP2 and HuNu (human nuclear antigen) staining shows the morphology of iGlut and neuron purity in iGlut-mAst co-cultures. (E-F) ISL2 ChIP-qPCR on day 30 iGlut-mAst co-cultures. n=3 replicates from one clone per line. (G-H) ISL2 siRNA knockdown in day-30 pure iGlut cultures. Samples of 72 hours post-siRNA transfection were used for qPCR. n=3 replicates from one clone per line. (I) Neuronal CLU mRNA level in isogenic iGlut-mAst co-cultures of different genotype of rs1532278 (human-specific CLU qPCR assay was used). n=6 per group (2-3 clones per line and 2-3 replicates for each clone). (J) Secreted CLU (sCLU) detected by ELISA from the supernatant of iGlut-mAst co-cultures. n=4 per group (2 clones per line, 2 replicates per clone). (K-L) Immunofluorescence staining of CLU in day 23-25 pure iGlut cultures. n=12 coverslips per group of two independent experiments (In total: 2 clones per line and 6 coverslips for each clone; and 5-6 cells per coverslip). (M) CLU mRNA level in the gray matter of postmortem brains of patients with AD and controls. Violin plots show data median and interquartile range, all other graphs show data mean±SEM. * p <0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Scale bars are indicated in each image.

Article Snippet: ELISA quantifications of human CLU (DCLU00, R&D system, specific for human CLU), human Aβ 1-40 (DAB140B, R&D system), and human Aβ 1-42 (DAB142, R&D system) were performed according to the vendors’ protocol.

Techniques: Binding Assay, Functional Assay, CRISPR, Staining, Knockdown, Transfection, Clone Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence

Journal: medRxiv

Article Title: Alzheimer’s disease protective allele of Clusterin modulates neuronal excitability through lipid-droplet-mediated neuron-glia communication

doi: 10.1101/2024.08.14.24312009

Figure Lengend Snippet: (A) Multiz alignment and phyloP conservation (470 mammals) around rs1532278 (from UCSC hg38 genome browser). (B) JASPAR predicted TF binding sites at rs1532278 and TF expression levels in iGlut (10/23 predicted TFs can be detected by RNA seq). CPM, counts per million reads. (C) DRGX ChIP-qPCR for iGlut-mAst co-cultures of CD07 line on day 30. n=3 per group (one clone with 3 replicates from the CD07 line). (D) CLU mRNA levels in iGlut pure cultures. n=6 per group (2-3 clones per line and 2-3 replicates for each clone). (E) sCLU levels detected by ELISA from the supernatant of iGlut pure cultures. n=4 per group (2 clones per line and 2 replicates for each clone). (F) CLU mRNA levels of mAst in iGlut-mAst co-cultures. n=4 per group (2 clones per line and 2 replicates for each clone). (G) Three qPCR primer sets that can capture the majority of CLU transcript isoforms (14/17) are used in (H) to detect CLU mRNA levels. Only iGlut-mAst co-cultures of the CD07 line was used. n=6 per group (3 replicates per clone for 2 clones). (I) rs1532278 did not alter the expression of SCARA3 , a CLU -adjacent gene, in iGlut (or when co-cultured with mAst) of the isogenic pairs of both CD05 and CD07 lines. n=10 per group (2 replicates per clone and 2-3 clones per line). Data = mean±SEM. * p <0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: ELISA quantifications of human CLU (DCLU00, R&D system, specific for human CLU), human Aβ 1-40 (DAB140B, R&D system), and human Aβ 1-42 (DAB142, R&D system) were performed according to the vendors’ protocol.

Techniques: Binding Assay, Expressing, RNA Sequencing Assay, Clone Assay, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: medRxiv

Article Title: Alzheimer’s disease protective allele of Clusterin modulates neuronal excitability through lipid-droplet-mediated neuron-glia communication

doi: 10.1101/2024.08.14.24312009

Figure Lengend Snippet: (A) Correlation analysis Salmon sorted iGlut data set to our previous published single-cell data sets iGlut (CD05 and CD07) and others published single cell data sets in postmortem brain with various cell types including ex-neuron, ih-neuron, microglia, astrocyte, oligodendrocytes, and OPC. Ast: astrocyte, ex: excitatory neuron, ih: inhibitory neuron, mic, microglia, Oli: oligodendrocyte, and OPC: oligodendrocyte progenitor cell. (B) MDS (Multidimensional scaling) analysis of all transcripts in iGlut and mAst, which are expressed in ≥75% of samples. (C) ELISA quantification of Aβ in the supernatants of iGlut-mAst co-cultures and iGlut pure cultures. n=4 per group (2 clones per line and 2 replicates for each clone). (D) GO Biological process enrichment in significantly downregulated gene sets in iGlut. Blue stars indicate neuron morphology-related pathways. (E) Confirmation of SYP , PSD-95 , and CLU expression levels in iGlut in RNA seq analysis. * p <0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: ELISA quantifications of human CLU (DCLU00, R&D system, specific for human CLU), human Aβ 1-40 (DAB140B, R&D system), and human Aβ 1-42 (DAB142, R&D system) were performed according to the vendors’ protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Clone Assay, Expressing, RNA Sequencing Assay