Journal: Cancers

Article Title: Multiparametric Characterization of the DSL-6A/C1 Pancreatic Cancer Model in Rats

doi: 10.3390/cancers16081535

Figure Lengend Snippet: List of antibodies used for the immunological analysis of the DSL-6A/C1 tumors.

Article Snippet: CD11a , PE , REA596 , Human cell line , Miltenyi , 130-109-171.

Techniques:

Journal: Channels

Article Title: Cone dystrophy and ectopic synaptogenesis in a Cacna1f loss of function model of congenital stationary night blindness (CSNB2A)

doi: 10.1080/19336950.2017.1401688

Figure Lengend Snippet: Primary antibodies used in this study.

Article Snippet: PKCα , Mouse , Purified Mouse Monoclonal IgG2a , 1:1000 , Cedarlane Labs, Burlington, ON , purified bovine brain PKCα (Clone MC5) , [ ] .

Techniques: Affinity Purification, CRAfT Assay, Purification, Recombinant

Journal: Science Advances

Article Title: The tumor suppressor adenomatous polyposis coli regulates T lymphocyte migration

doi: 10.1126/sciadv.abl5942

Figure Lengend Snippet: Antibodies used for flow cytometry.

Article Snippet: APC anti-human CD11a , Mouse IgG1 , Miltenyi Biotec 130-127-294 , 1:50.

Techniques: Cytometry

Journal: bioRxiv

Article Title: Mouse and human microglial phenotypes in Alzheimer’s disease are controlled by amyloid plaque phagocytosis through Hif1α

doi: 10.1101/639054

Figure Lengend Snippet: a , Schematic of the methodology employed in this study . b , Representative immunofluorescence image of the hippocampus of WT and 5xFAD mice injected with Methoxy-XO4 and stained with Iba1 (AlexaFluor 488, n =6 animals per genotype), scale bar=250 μm, inset 50 μm c , Representative FACS plot (from n =12-19 animals per genotype and age group) showing that XO4 + microglia are present in 6m 5xFAD plaque-affected regions (top panels). d , left, the percentage of XO4 + microglia isolated from plaque-affected regions in 1, 4, and 6m old WT and 5xFAD mice, ( n = 12-19 per genotype and age group; male and female mice pooled) and right, the percentage of XO4 + microglia isolated from plaque-affected and non-affected regions in 6m old male and female WT and 5xFAD mice ( n = 6-8 per genotype), expressed as mean ± SEM. e , PCA of bulk RNA-seq. Cx, Cortex; Cb, Cerebellum f, g Gene cytometry plots showing genes that are differentially expressed between XO4 + and XO4 − microglia and/or genes that are differentially expressed between old (4, 6 month) and young (1 month) microglia. Gene scores are calculated as the product of the log fold change and –log 10 (FDR). Example genes in each quadrant are labelled in red (upregulated over time or phagocytosis) or blue (downregulated). h i , Venn diagram showing the overlap between genes whose expression levels could be explained by the age, region and XO4 covariate as well as GO and KEGG terms associated with XO4 covariate genes. h ii , table showing the 21 core microglial neurodegeneration signature genes and their direction of differential expression in DAM , CD11c + , MGnD and XO4 + microglia. i , heat map of targeted LC-SWATH-MS analysis of detected peptides within DEGs in n =3-5 biological replicates of WT (blue), XO4 − 5xFAD (orange) and XO4 + 5xFAD (green) microglia. j , comparison of RNA and protein expression for selected genes, and quantitation of a tryptic peptide in Aβ in microglia. Data are expressed as mean ± SEM log fold change compared to WT microglia, normalized relative to peptides in Supplementary table 2. p values in d and j were calculated by one-way ANOVA using Tukey’s multiple comparison test.

Article Snippet: The cell pellet was then stained with antibodies to microglial cell surface markers (CD11b-BV650, 1:200 Biolegend, #141723; CD45-BV786, 1:200, BD Biosciences #564225; CX3CR1-FITC, 1:100, Biolegend, #149019; CD11a, 1:20, BD Biosciences, #558191, TREM2-APC, 1:10, R&D Systems, #FAB17291N; CD33-PE, 1:20, eBioscience, #12-0331-82; CD115-BV711, 1:40, Biolegend, #135515) for isolation using the FACSAria™ III cell sorter.

Techniques: Immunofluorescence, Injection, Staining, Isolation, RNA Sequencing, Cytometry, Expressing, Comparison, Quantitation Assay

Journal: bioRxiv

Article Title: Mouse and human microglial phenotypes in Alzheimer’s disease are controlled by amyloid plaque phagocytosis through Hif1α

doi: 10.1101/639054

Figure Lengend Snippet: a , Dimensionality reduction representation (viSNE, representative of n =3 mice per genotype) of myeloid cells isolated from WT (top) and 5xFAD (bottom) 6m male mice. Microglia (CD11b + CD45 lo CX3CR1 + ) are colored for expression of CD11b, CD45, CX3CR1, Methoxy-XO4, CD115, CD33 and TREM2, whereas remaining myeloid cells are grayscale for clarity. b , PCA of 893 single cells x 1671 feature genes showing the distribution of cells from each FACS-sorted sample. c , PCA plot of single microglia colored by SC3 clusters and composition of automated clusters as a percentage of sequenced FACS-sorted cell populations. d . PCA plots for single microglia colored by expression of selected ageing microglia genes (i-ii), homeostatic (iii) and XO4 + signature genes (iv-v). e, f Diffusion maps pseudotime analysis of microglial populations ordered by their expression of e , ageing DEGs (6m WT v 24m WT, 42 DEGs) or f , phagocytic DEGs (6m 5xFAD XO4 − v 6m 5xFAD XO4 + , 474 DEGs) g , scatter plot showing the relationship between ageing and phagocytosing pseudotime in individual cells, and the density of cells at each point during the ageing (bottom) and phagocytosing (left) trajectories. h , Hierarchical clustering and heat map showing expression of the top 50 DEGs across the 4 SC3 clusters.

Article Snippet: The cell pellet was then stained with antibodies to microglial cell surface markers (CD11b-BV650, 1:200 Biolegend, #141723; CD45-BV786, 1:200, BD Biosciences #564225; CX3CR1-FITC, 1:100, Biolegend, #149019; CD11a, 1:20, BD Biosciences, #558191, TREM2-APC, 1:10, R&D Systems, #FAB17291N; CD33-PE, 1:20, eBioscience, #12-0331-82; CD115-BV711, 1:40, Biolegend, #135515) for isolation using the FACSAria™ III cell sorter.

Techniques: Isolation, Expressing, Diffusion-based Assay

Journal: bioRxiv

Article Title: Mouse and human microglial phenotypes in Alzheimer’s disease are controlled by amyloid plaque phagocytosis through Hif1α

doi: 10.1101/639054

Figure Lengend Snippet: a-c UMAP projection of single microglia nuclei from control and AD patient frontal cortex, cases ( n =172 nuclei) and controls ( n =277 nuclei). The microglial population was determined by similarity to known microglial markers . The UMAP projection is colored by a , disease diagnosis b , XO4 + score c , or ageing signature score. Diffusion maps pseudotime analysis of microglial populations ordered by their expression of d , XO4 + DEGs (taking top 10% of respective DE genes regardless of overlap with aging DEGs, 167 DEGs between 5xFAD XO4 + and XO4 − mice) or e , ageing DEGs (top 10% or 167 DEGs between 24M WT and 6M WT mice). f , scatter plot showing the relationship between ageing and XO4 + pseudotime in individual cells and the density of cells at each point during the ageing (left) and XO4 + (bottom) trajectories. g , UMAP projection of single human microglia colored by expression of selected cluster specific-genes. h Hierarchical clustering and heat map showing expression of the overlapping DEGs in each of the 4 mouse microglia clusters with the DEGs between human control and AD microglia. The mouse-human concordance on the direction of change between control and AD (human) or WT and XO4 + populations is shown for each gene ( p =0.000641). i , SCENIC regulon analysis showing that Hif1a and Elf3 are predicted to control the XO4 + gene regulatory network. j , UMAP projection of single human microglia colored by HIF1A regulon activity. k , Fluorescently labeled synaptosome internalization by primary microglia transfected with GFP-tagged inducible HIF1A and/or ELF3 overexpression constructs. The data are presented as mean ± SEM and show the difference in synaptosome internalization between GFP + and GFP − (non-transfected) cells tested from within the same well.

Article Snippet: The cell pellet was then stained with antibodies to microglial cell surface markers (CD11b-BV650, 1:200 Biolegend, #141723; CD45-BV786, 1:200, BD Biosciences #564225; CX3CR1-FITC, 1:100, Biolegend, #149019; CD11a, 1:20, BD Biosciences, #558191, TREM2-APC, 1:10, R&D Systems, #FAB17291N; CD33-PE, 1:20, eBioscience, #12-0331-82; CD115-BV711, 1:40, Biolegend, #135515) for isolation using the FACSAria™ III cell sorter.

Techniques: Control, Diffusion-based Assay, Expressing, Activity Assay, Labeling, Transfection, Over Expression, Construct

Journal: Cancer Cell

Article Title: Multimodal stimulation screens reveal unique and shared genes limiting T cell fitness

doi: 10.1016/j.ccell.2024.02.016

Figure Lengend Snippet: Loss of Icam1 -mediated homotypic T cell interactions amplifies CD8 T cell expansion and improves effector functions shortly after TCR stimulation (A) Overlapping genes from top 50 hits from each screen, genes are ranked by average effect size. (B) STRING protein-protein interaction analysis of shared targets from the two boxes in (A) (32 genes). Interactions include direct (physical) and indirect (functional) associations. (C) Kaplan-Meier OS curves of patients receiving TIL therapy , (Besser cohort) with high or low ICAM1 expression in TIL products. Significance calculated with regular log rank test. (D) Microscopy images of indicated T cells 24 h after CD3 stimulation (n = 7 biological replicates). (E) Viable Ctrl and Icam1 -KO T cell counts under indicated stimulation conditions as in the screens, analyzed with Mann-Whitney test (n = 4–7 biological replicates). (F) Viable B16.OVA cells after 4 days co-culture with indicated T cells, analyzed with Mann-Whitney test (n = 4 biological replicates). (G) Transcriptomic profiling heatmap of indicated T cells with or without 24 h-CD3 stimulation, showing significantly (p value <0.001) differentially expressed genes ( Table S3 ). (H) Proteomic STRING enrichment analysis of differentially expressed proteins comparing Icam1 -KO with Ctrl T cells after 24 h-CD3 stimulation, showing top enriched GO biological process (ranked by enrichment strength (Log 10 (observed/expected)) with FDR < 0.1 ( Table S3 ). (I) Flow cytometry-based cytokine bead array showing cytokines released in the culture medium of indicated T cells after 24 h-CD3 stimulation, analyzed with Mann-Whitney test (n = 7 biological replicates). (J) Viable cell counts of Ctrl or Icam1 -KO T cells ectopically expressing wild type (wt) or mutated ICAM1 (lacking the intracellular domain, dcyt). Cell count was assessed 4 days after CD3 stimulation; analyzed with one-way ANOVA with Holm-Sidak’s multiple comparisons test (n = 3 biological replicates). OE: overexpression. (K) Viable cell counts of Ctrl or ICAM1/2/3 -KO human T cells 1 week after 24 h-CD3 stimulation, analyzed with Mann-Whitney test (n = 5 biological replicates). (L) Viable cell counts of human CD8 cells 1week after CD3 stimulation with or without CD54 (ICAM1) or CD11a (LFA1) blocking antibodies, analyzed with Kruskal-Wallis test with Dunn’s post-hoc test (n = 3 biological replicates). Error bars indicate SD. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: T cell reactivation was performed in the presence of CD11a antibody (1 μg/ml, clone R7-1, BioXCell), CD54 (1 μg/ml, clone R6-5-D6, BioXCell) antibody, CD178/FasL (10 μg/ml, Clone MFL3 (RUO), BD Pharmingen) or respective isotype controls (1 μg/ml, mouse IgG2a, clone C1.18.4 and mouse IgG1, clone MOPC-21, both BioXCell; 10 μg/ml, Armenian Hamster IgG1, κ, BD Pharmingen).

Techniques: Functional Assay, Expressing, Microscopy, MANN-WHITNEY, Co-Culture Assay, Flow Cytometry, Cell Counting, Over Expression, Blocking Assay

Journal: Cancer Cell

Article Title: Multimodal stimulation screens reveal unique and shared genes limiting T cell fitness

doi: 10.1016/j.ccell.2024.02.016

Figure Lengend Snippet:

Article Snippet: T cell reactivation was performed in the presence of CD11a antibody (1 μg/ml, clone R7-1, BioXCell), CD54 (1 μg/ml, clone R6-5-D6, BioXCell) antibody, CD178/FasL (10 μg/ml, Clone MFL3 (RUO), BD Pharmingen) or respective isotype controls (1 μg/ml, mouse IgG2a, clone C1.18.4 and mouse IgG1, clone MOPC-21, both BioXCell; 10 μg/ml, Armenian Hamster IgG1, κ, BD Pharmingen).

Techniques: Purification, Blocking Assay, Control, Functional Assay, Recombinant, Staining, Cell Culture, Isolation, Binding Assay, CRISPR, Sequencing, RNA Sequencing, Software