Journal: Nature Communications

Article Title: Microglial dopamine receptor elimination defines sex-specific nucleus accumbens development and social behavior in adolescent rats

doi: 10.1038/s41467-018-06118-z

Figure Lengend Snippet: C3–C3R interactions mediate developmentally-typical D1r elimination in vivo in males, but not females. Neutrophil inhibitor factor (NIF), a peptide that binds specifically to the CD11b subunit of C3 receptors (C3Rs), was assessed for its efficacy in reducing microglial phagocytic activity ex vivo. a In microglia isolated from whole brain, 60 ng, but not 120 ng NIF inhibited microglial phagocytosis of fluorescent beads (Table ). Representative images precede histograms; scale bar equals 20 µm. n = 4, with 2 replications/condition. b Microglia were isolated from whole brain and incubated with 60 ng NIF, and then immunocytochemically assessed for NIF and CD11b at 30, 60, and 120 min. Closed arrow heads indicate NIF (green) immunoreactivity. Scale bar equals 10 µm. There was no change in c NIF or (Table ) d CD11b (Table ) immunoreactivity over 2 h, suggesting that NIF was not causing the degradation of its receptor. n = 3; 2 replications/condition. To determine if developmental D1r downregulation requires C3–C3R interactions, NIF or Vehicle was microinjected in the NAc at e P30 in males and g P22 in females (both represent sex-specific ages prior to D1r downregulation), and then tissue was assessed at P38 and P30, respectively, an age at which D1rs should be developmentally downregulated. f In males, NIF-treated hemispheres exhibited significantly more (~25%) D1r immunoreactivity than within-animal vehicle-treated hemispheres (Table ). h In females, NIF-treated hemispheres exhibited the same D1r immunoreactivity as within-animal vehicle-treated hemispheres (Table ). Representative images precede histograms; scale bars equal 100 µm. n = 4/sex with counterbalanced injections. For ex vivo experiments, data were analyzed with one-way ANOVAs and Holm-Sidak’s post-hoc comparisons. For in vivo experiments, D1r data from 4–7 different sections per animal were averaged and then calculated as a percentage of within-animal vehicle control levels. Data were analyzed with 2-tailed one-sample t -tests. Histograms portray the mean ± SEM with individual data points overlaid. Significant post-hoc Holm-Sidak t -tests ( a – d ) and one-sample t -tests from 100 ( f , h ) ( p < 0.05) comparisons are delineated with an asterisk. All statistics are in Table

Article Snippet: Resulting cell bodies were incubated with anti-rat CD11b (i.e., microglia-specific) magnetic beads (Miltenyi Biotec #130–105–634; Auburn, CA) and then separated from the CD11b- population via magnetic columns.

Techniques: In Vivo, Activity Assay, Ex Vivo, Isolation, Incubation, Control

Journal: Nature Communications

Article Title: Microglial dopamine receptor elimination defines sex-specific nucleus accumbens development and social behavior in adolescent rats

doi: 10.1038/s41467-018-06118-z

Figure Lengend Snippet: Detailed statistics corresponding with Fig. 3

Article Snippet: Resulting cell bodies were incubated with anti-rat CD11b (i.e., microglia-specific) magnetic beads (Miltenyi Biotec #130–105–634; Auburn, CA) and then separated from the CD11b- population via magnetic columns.

Techniques: Comparison, Ex Vivo, In Vivo, Injection

Journal: bioRxiv

Article Title: Inhibition of Retinoic Acid Signaling in Proximal Tubular Epithelial cells Protects against Acute Kidney Injury by Enhancing Kim-1-dependent Efferocytosis

doi: 10.1101/2023.06.15.545113

Figure Lengend Snippet: PTEC DN-RAR mice underwent rhabdo or bilateral IRI-AKI, and kidneys harvested after 3 days. A-D, Increased F4/80 staining in the kidney after rhabdo- and IRI-AKI. A/B, F4/80 staining in uninjured mice (Ctrl) and after rhabdo-AKI (Rh-AKI). Area staining with F4/80 in the OSOM, and representative images showing F4/80, Sox9, and LTL staining. Left hand panels show F4/80 staining is largely restricted to the OSOM. Right hand panels show higher magnification of the OSOM. C/D, F4/80 staining after bilateral IRI-AKI. Quantification, and images showing F4/80 and LTL staining in the OSOM. Results expressed as means +/- SEM, individual data points shown. A/C, 1-way ANOVA, if p <0.05, q values shown for between group comparisons. Scale bars=100μM. E-F, Decreased expression of inflammatory markers in CD11B+ cells after IRI-AKI. PTEC DN-RAR mice underwent bilateral IRI-AKI, and bulk RNA seq performed on renal CD11B + cells 3 days after injury. E, Gene set enrichment analysis (GSEA) of downregulated genes using Gene Ontology (GO) datasets. F, GSEA for validated pro-inflammatory (“M1”), and anti-inflammatory (“M2”) gene sets in the RNA seq data. Set size=no. of genes from each gene set that are represented. NES=normalized expression score for the gene set sizes. G/H, Volcano plots showing fold change in expression of core enrichment gene from the CD11B+ bulk RNA seq dataset. Dotted line indicates p<0.05.

Article Snippet: Cells were then incubated with anti-mouse CD11b MACS beads (Miltenyi Biotech, 130-126-725) for 15 minutes, and CD11b+ cells separated by loading cells into MACS separator.

Techniques: Staining, IF-P, Expressing, RNA Sequencing

Journal: bioRxiv

Article Title: Inhibition of Retinoic Acid Signaling in Proximal Tubular Epithelial cells Protects against Acute Kidney Injury by Enhancing Kim-1-dependent Efferocytosis

doi: 10.1101/2023.06.15.545113

Figure Lengend Snippet: PTEC DN-RAR mice underwent bilateral IRI-AKI, and bulk RNA seq was performed on CD11B + cells from 3 PEPCK Cre+ and 3 Cre- mouse kidneys 3 days after injury. PCA shows clear separation of Cre+ and Cre- datasets in the first dimension, which accounts for 57% of the variation.

Article Snippet: Cells were then incubated with anti-mouse CD11b MACS beads (Miltenyi Biotech, 130-126-725) for 15 minutes, and CD11b+ cells separated by loading cells into MACS separator.

Techniques: RNA Sequencing

Journal: bioRxiv

Article Title: Inhibition of Retinoic Acid Signaling in Proximal Tubular Epithelial cells Protects against Acute Kidney Injury by Enhancing Kim-1-dependent Efferocytosis

doi: 10.1101/2023.06.15.545113

Figure Lengend Snippet: A, CD45 + cells: CD45 vs SSC-A; B, CD45 + single cells: FSC-H vs FSC-A, selected upper line followed by SSC-H vs. SSC-A select upper line; C, Single, CD45 + live cells: DAPI - vs. CD45 + ; D, Exclude CD45 + granulocytes: Ly6G + (granulocytes) and Ly6G - cells (non-granulocytes); E, F4/80 expression in CD11B + ; Ly6G - cells: F4/80 - ; Intermediate (int), 2); high (hi) (3); F-H, Ly6C expression: F, F4/80 - : Ly6C - (1), Int (2), high (3); G, F4/80 Int : Ly6C - (1), Int (2), high (3); H, F4/80 Hi : Ly6C - (1), Int (2), high (3); I-K, MHC-II expression: I, F4/80 - : MHC -- (1), Int (2), high (3); J, F4/80 Int : MHC-II - (1), Int (2), high (3); K, F4/80 Hi : MHC-II - (1), Int (2), high (3).

Article Snippet: Cells were then incubated with anti-mouse CD11b MACS beads (Miltenyi Biotech, 130-126-725) for 15 minutes, and CD11b+ cells separated by loading cells into MACS separator.

Techniques: Expressing

Journal: bioRxiv

Article Title: Inhibition of Retinoic Acid Signaling in Proximal Tubular Epithelial cells Protects against Acute Kidney Injury by Enhancing Kim-1-dependent Efferocytosis

doi: 10.1101/2023.06.15.545113

Figure Lengend Snippet: Kidneys were harvested from 5 PEPCK Cre+ and 10 Cre- uninjured mice, and from 9 PEPCK Cre+ and 10 Cre- mice 3 days after bilateral IRI-AKI. Tissue was digested and homogenized and evaluated by flow cytometry using antibodies and gating strategy illustrated in . A, Total numbers of live, single CD45+ cells acquired; B, CD11B+ cells; and C, non-granulocytes (CD11B + ; Ly6G - ). Results expressed as means +/- SEM, with individual data points shown. 1-way ANOVA was used to compare between groups, and if p <0.05, q values shown for between group comparisons, corrected for repeated testing.

Article Snippet: Cells were then incubated with anti-mouse CD11b MACS beads (Miltenyi Biotech, 130-126-725) for 15 minutes, and CD11b+ cells separated by loading cells into MACS separator.

Techniques: Flow Cytometry, IF-P

Journal: bioRxiv

Article Title: Inhibition of Retinoic Acid Signaling in Proximal Tubular Epithelial cells Protects against Acute Kidney Injury by Enhancing Kim-1-dependent Efferocytosis

doi: 10.1101/2023.06.15.545113

Figure Lengend Snippet: Kidneys were harvested from PTEC DN RAR mice 3 days after bilateral IRI-AKI. Tissue was digested, homogenized, and evaluated by flow cytometry. A/B, F4/80 hi cells (kidney resident macrophages). A, F4/80 hi cells as the % of gated CD11B + CD45 + Ly6G - cells. B, CD11B and F4/80 expression charts in PEPCK Cre+ and Cre- mice after IRI-AKI (% in gated area indicated with *). C-F, Ly6C hi cells (inflammatory monocyte/macrophages) . C, F4/80 - (infiltrating monocytes) Ly6C hi cells, and E, F4/80 int (bone marrow derived macrophages) Ly6C hi cells, as the % of gated F4/80 - and F4/80 int cells, respectively. D/F, CD11B and Ly6C expression charts. G/H, CD206/mannose receptor expression (an M2 activated macrophage marker) . G, CD206 + cells as the % of gated F4/80 hi cells. H, CD11B and CD206 expression charts. Results are expressed as means +/- SEM, with individual datapoints shown. 1-way ANOVA was used to compare between groups, and if p <0.05, q values shown for between group comparisons, corrected for repeated testing.

Article Snippet: Cells were then incubated with anti-mouse CD11b MACS beads (Miltenyi Biotech, 130-126-725) for 15 minutes, and CD11b+ cells separated by loading cells into MACS separator.

Techniques: Flow Cytometry, Expressing, Derivative Assay, Marker, IF-P

Journal: bioRxiv

Article Title: Inhibition of Retinoic Acid Signaling in Proximal Tubular Epithelial cells Protects against Acute Kidney Injury by Enhancing Kim-1-dependent Efferocytosis

doi: 10.1101/2023.06.15.545113

Figure Lengend Snippet: Kidneys were harvested from PTEC DN RAR mice, uninjured and 3 days after bilateral IRI AKI. Tissue was digested, homogenized, and evaluated by flow cytometry. A/B, MHC-II hi F4/80 hi cells (kidney resident macrophages). A, MHC-II hi as the % of gated F4/80 hi cells. B, Representative CD11B and MHC-II expression chart in PEPCK Cre+ and Cre- mice after IRI-AKI (% in gated area indicated with *). C/D, EdU + F4/80 hi cells (proliferating kidney resident macrophages). C, EdU labeled cells as the % of gated F4/80 hi cells. D, Representative CD11B (Y axis) and EdU (X-axis) staining charts. E/F, Immunofluorescence staining with monocyte and neutrophil markers, Gr-1 and MPO. Kidneys were harvested from PTEC DN RAR mice 3 days after bilateral IRI-AKI and co-labeled with LTL and either Gr-1 (a marker of activated monocytes and neutrophils), or MPO (a marker of neutrophils) antibodies. G/H, G, Quantification of the Gr-1 and MPO+ cells as the % of the total cells counted. Results are expressed as means +/- SEM, with individual datapoints shown. 1-way ANOVA used to compare between groups, and if p <0.05, q values shown for between group comparisons, corrected for repeated testing.

Article Snippet: Cells were then incubated with anti-mouse CD11b MACS beads (Miltenyi Biotech, 130-126-725) for 15 minutes, and CD11b+ cells separated by loading cells into MACS separator.

Techniques: Flow Cytometry, Expressing, Labeling, Staining, Immunofluorescence, Marker, IF-P