Journal: Cell reports

Article Title: Activation of GPR44 decreases severity of myeloid leukemia via specific targeting of leukemia initiating stem cells

doi: 10.1016/j.celrep.2023.112794

Figure Lengend Snippet:

Article Snippet: Mouse anti-mouse CD45.1 (FITC) , Miltenyi Biotec , Cat# 130–124-211; RRID: AB_2857674.

Techniques: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay, Staining, Modification, Saline, Concentration Assay, Over Expression, Protein Extraction, Membrane, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Protease Inhibitor, CCK-8 Assay, Reverse Transcription, Selection, Plasmid Preparation, Knock-Out, Software, Real-time Polymerase Chain Reaction, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Multifocal Cerebral Microinfarcts Modulate Early Alzheimer’s Disease Pathology in a Sex-Dependent Manner

doi: 10.3389/fimmu.2021.813536

Figure Lengend Snippet: Dynamics of circulating monocytes are modulated upon multifocal microinfarction. (A) Gating strategy used to discriminate the circulating monocytes (CD11b + LyC6 + ) in leukocytes (CD45 + ) and the subsequent distribution of the inflammatory monocytes (Ly6C high ), patrolling monocytes (Ly6C low ) and neutrophils (Ly6G high ). (B) Flow cytometry analysis shows that the frequency of total monocytes in the blood circulation increases in MO-operated male WT mice while it remains unchanged in MO-operated male APP/PS1 mice compared to sham-operated male mice and decreases in MO-operated male APP/PS1 mice compared to MO-operated male WT mice. (C) Flow cytometry analysis shows that the frequency of Ly6C low monocytes decreases in the blood circulation of MO-operated male WT mice compared to sham-operated WT mice. (D) Flow cytometry analysis shows that the frequency of Ly6C int monocytes increases in the blood circulation of MO-operated male WT mice as well as MO-operated male APP/PS1 mice compared to sham-operated WT mice. (E) Flow cytometry analysis shows that the frequency of inflammatory Ly6C high monocytes decreases in the blood circulation of MO-operated male WT mice as well as MO-operated male APP/PS1 mice compared to sham-operated male mice. (F) Flow cytometry analysis shows that similarly to males, the frequency of total monocytes in the blood circulation increases in MO-operated female WT mice while it remains unchanged in MO-operated female APP/PS1 mice compared to sham-operated female mice and decreases in MO-operated female APP/PS1 mice compared to MO-operated female WT mice. (G) Flow cytometry analysis indicates that the frequency of Ly6C low monocytes increases in MO-operated female WT mice, while it decreases in the MO-operated female APP/PS1 mice compared to sham-operated female mice. (H) Flow cytometry analysis indicates that the frequency of Ly6C int monocytes increases in the blood circulation of MO-operated female WT mice and to a lesser extent MO-operated female APP/PS1 mice. (I) Flow cytometry analysis indicates that the frequency of Ly6C high monocytes is decreased in the blood circulation of MO-operated female WT mice as well as MO-operated female APP/PS1 mice compared to sham-operated female mice. (J) Flow cytometry analysis shows that the frequency of Ly6G neutrophils increases in MO-operated male WT mice compared to sham-operated male WT mice but to a lesser extent in MO-operated male APP/PS1 mice compared to sham-operated APP/PS1 male mice. (K) Flow cytometry analysis shows that the frequency of Ly6G neutrophils similarly increases in the blood circulation of MO-operated female WT mice compared to sham-operated female WT mice, whereas it remains unchanged in MO-operated female APP/PS1 mice compared to sham-operated female APP/PS1 mice. Data are mean ± SEM (n = 5-10 animals/group). *P < 0.05/**P < 0.01/****P < 0.01 compared with sham- and MO-operated male and female WT and APP/PS1 mice (two-tailed unpaired t-test).

Article Snippet: The following primary antibodies were used; anti-Aβ (6E10) (1:1000; mouse anti-human, Biolegend, 8030001), anti-ionized calcium binding adaptor molecule (IBA)-1 (1:1000, rabbit anti-mouse, WAKO, 019-19741), anti-cluster of differentiation (CD)-68 (1:1000, rat anti-mouse, Bio-Rad, Hercules, CA, USA, MCA1957) and anti-CD45 (1:500, rat anti-mouse, BD bioscience, 55376) and anti-nuclear receptor subfamily 4 group A member 1 (Nr4A1 or Nurr77) (1:250, rabbit, Abcam, ab13851).

Techniques: Flow Cytometry, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Multifocal Cerebral Microinfarcts Modulate Early Alzheimer’s Disease Pathology in a Sex-Dependent Manner

doi: 10.3389/fimmu.2021.813536

Figure Lengend Snippet: Infiltrated phagocytic monocytes are recruited to the lesion sites and Aβ plaques. (A) A tile representing a coronal brain section of MO-operated male APP/PS1/CX3CR1 GFP/+ mouse immunolabeled with CD45 (i.e. infiltrating cells, blue) and IBA1 (i.e. microglia; red) showing (A) the presence of CX3CR1 GFP/+ expressing Nurr77 + , highlighting their monocytic origin, at lesion site 1 week post-microinfarct induction. (B) A tile representing a coronal brain section of MO-operated male APP/PS1/CX3CR1 GFP/+ mouse immunolabeled with CD45 (blue) and 6E10 (Aβ plaques; red) showing (B’) the presence of CD45 + /CX3CR1 GFP/+ cells at the lesion site and (B’’) CD45 + /IBA1 + /CX3CR1 GFP/+ cells recruited to Aβ plaques. Scale bar = 20 µm (A) , 1000 µm (B) , 50 µm (B’) and 25 µm (B”) .

Article Snippet: The following primary antibodies were used; anti-Aβ (6E10) (1:1000; mouse anti-human, Biolegend, 8030001), anti-ionized calcium binding adaptor molecule (IBA)-1 (1:1000, rabbit anti-mouse, WAKO, 019-19741), anti-cluster of differentiation (CD)-68 (1:1000, rat anti-mouse, Bio-Rad, Hercules, CA, USA, MCA1957) and anti-CD45 (1:500, rat anti-mouse, BD bioscience, 55376) and anti-nuclear receptor subfamily 4 group A member 1 (Nr4A1 or Nurr77) (1:250, rabbit, Abcam, ab13851).

Techniques: Immunolabeling, Expressing

Journal: Cell Proliferation

Article Title: p75NTR −/− mice exhibit an alveolar bone loss phenotype and inhibited PI3K/Akt/β‐catenin pathway

doi: 10.1111/cpr.12800

Figure Lengend Snippet: P75NTR −/− EMSCs exhibit decreased osteogenic differentiation capacity compared to WT EMSCs. (A) The p75NTR and mesenchymal stem cell surface markers (CD14, CD90, CD146 and CD166) and hematopoietic markers (CD45) were detected on WT and p75NTR −/− EMSCs by the Flow cytometry. (B) The third passage (P3) cells of E12.5d WT and p75NTR −/− EMSCs. Scale bar represents 50 μm. (C) Representative images of colonies formed by E12.5d WT and p75NTR −/− EMSCs and the analysis of colony formation. (D) The proliferation ratio of E12.5d WT and p75NTR −/− EMSCs was assessed by CCK‐8. WT and p75NTR −/− EMSCs were induced with osteogenic induction medium. On days 7, 14 and 21, the (E) protein levels of Runx2, Col1 and β‐catenin were detected by Western blot and (F) grayscale analysis was performed and the levels of the indicated proteins are expressed relative to the levels of GAPDH. (G) mRNA levels of Runx2, Col1 and β‐catenin were detected by real‐time PCR, respectively, using GAPDH as control. (H) On day 7, ALP staining was used to detect their potential of differential mineralization. On day 21, Alizarin Red staining was used to detect their mineralized nodules. Scale bar represents 50 μm. KO represent p75NTR −/− . Data are shown as mean ± SD from three independent experiments. * P < .05, ** P < .01

Article Snippet: The primary antibodies including anti‐mouse CD14, anti‐mouse CD90, anti‐mouse CD146, anti‐mouse CD166, anti‐mouse CD45 (1:100; Santa Cruz, USA) and anti‐mouse p75NTR‐FITC (1:100; Abcam, UK) were used to identify EMSCs.

Techniques: Flow Cytometry, CCK-8 Assay, Western Blot, Real-time Polymerase Chain Reaction, Control, Staining

Journal: Aging Cell

Article Title: Compromised CD8+ T cell immunity in the aged brain increases severity of neurotropic coronavirus infection and postinfectious cognitive impairment

doi: 10.1111/acel.14409

Figure Lengend Snippet: Aging limits T RM establishment within the brain. 8 week. adult or 18 months aged C57BL/6 mice were inoculated with 10 3 pfu MHV‐A59 i.n., CD8 + T cells harvested from the spleen 7 DPI and then adoptively transferred intravenously (i.v.) via the tail vein into either 8 week. or 18 months old animals that were infected with (a,b) 10 4 pfu or (c,d) 10 3 pfu MHV‐A59 1 day prior. (a, c) Survival and (b, d) weight change were monitored for 30 DPI. (e) Representative flow cytometry plots of lymphocytes stained for CD8, then CD45.1 and CD45.2 to distinguish between host and donor cells isolated from the brains of 8 week. or 18 months old animals at 30 DPI following infection with 10 3 pfu MHV‐A59 and adoptive transfer of 8 week. or 18 months cells as indicated. (f) Frequency and (g) total number of total CD8 + cells and total transferred cells. (h) Representative flow cytometry histograms of CD103 expression by CD8 + T cells present in the brain of 8 week. or 18 months animals 30 DPI following infection and adoptive transfer as described. (i) Quantification of CD103 MFI by host or donor cells within the same host. Data are representative of 1 independent experiment with each data point representing an individual animal. Survival assessed by Log‐rank Mantel‐Cox assessment, weight change and flow cytometry assessment conducted according to two‐way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Tetramer staining was carried out at room temperature for 20 min in conjugation with other surface staining antibodies: CD8a (53–6.7, APC‐Cy7), CD4 (RM4‐5, APC), CD44 (IM7, PE‐Cy7), CD45 (30‐F11, APC), CD45.1 (20–0453, APC), CD45.2 (60–0454, PE‐Cy‐7), CD11b (M1/70, FITC), P2RY12 (S16007D, PE), CD68 (FA‐11, BV‐421), MHC II (M5/114.15.2, PerCP‐Cy5.5), obtained from Cytek Biosciences or eBioscience, then washed with 1× PBS and fixed with 2% paraformaldehyde (PFA).

Techniques: Infection, Flow Cytometry, Staining, Isolation, Adoptive Transfer Assay, Expressing

Journal: Aging Cell

Article Title: Compromised CD8+ T cell immunity in the aged brain increases severity of neurotropic coronavirus infection and postinfectious cognitive impairment

doi: 10.1111/acel.14409

Figure Lengend Snippet: CD8 + T cells mediate neuronal apoptosis following MHV‐A59 infection. (a) Representative IHC of DAPI, NeuN, and CD8a in the DG and cortex of 8 week or 18 months old animals at (a) 12 DPI and (b) 30 DPI. (c) Representative flow cytometry plots of CD45 + CD8 + cells stained for IFN‐γ isolated from the brains of 8 week or 18 months old, mock or MHV‐A59 infected animals at 30 DPI following 4 h. PMA/ionomycin stimulation. (d) Quantification of percent CD8 + CD44 + cells positive for IFN‐γ. (e) Representative immunocytochemical (ICC) staining for NeuN and TUNEL of primary cortical neurons following no treatment or infection with MHV‐A59 MOI 0.5, with or without coculture with CD8 + T cells purified from the spleen of an 8 week old animal at 7 DPI with 10 3 pfu MHV‐A59. Quantification of ICC images: (f) total number NeuN + neuronal nuclei, (g) total number TUNEL + NeuN + neuronal nuclei, (h) proportion of TUNEL + NeuN + neuronal nuclei of total NeuN + neuronal nuclei present. (i) Representative ICC of NeuN and TUNEL staining of primary embryonic cortical neurons following no treatment or infection with MHV‐A59 MOI 0.5, with or without naïve or PMA/ionomycin stimulated CD8 + T cells purified from the spleen of an 8 week old uninfected animal. Quantification of ICC images: (j) total number NeuN + neuronal nuclei, (k) total number TUNEL + NeuN + neuronal nuclei, and (l) proportion of TUNEL + NeuN + neuronal nuclei of total NeuN + neuronal nuclei present. Data are representative of three independent experiments with each data point representing an individual sample. All images were taken at 40X magnification and scale bar = 100 μm. Three images were captured per sample and averaged. NeuN + and TUNEL + NeuN + neuronal nuclei quantified in ImageJ software using the Cell Counter plugin. Statistics according to unpaired one‐way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Tetramer staining was carried out at room temperature for 20 min in conjugation with other surface staining antibodies: CD8a (53–6.7, APC‐Cy7), CD4 (RM4‐5, APC), CD44 (IM7, PE‐Cy7), CD45 (30‐F11, APC), CD45.1 (20–0453, APC), CD45.2 (60–0454, PE‐Cy‐7), CD11b (M1/70, FITC), P2RY12 (S16007D, PE), CD68 (FA‐11, BV‐421), MHC II (M5/114.15.2, PerCP‐Cy5.5), obtained from Cytek Biosciences or eBioscience, then washed with 1× PBS and fixed with 2% paraformaldehyde (PFA).

Techniques: Infection, Flow Cytometry, Staining, Isolation, TUNEL Assay, Purification, Software

Journal: Journal of neuroinflammation

Article Title: Treatment with gelsolin reduces brain inflammation and apoptotic signaling in mice following thermal injury.

doi: 10.1186/1742-2094-8-118

Figure Lengend Snippet: Figure 8 Gelsolin affects migration of myeloid-derived cells into brain. CD45+ cells from gelsolin-treated mice 8 h postburn (A) and quantification of infiltrating CD45+ (B) cells in 10 high power fields (HPF) of the periventricle region following gelsolin treatment. Gelsolin positive cells were seen in medial habenular nucleus (MHb), stria medullaris (sm), hippocampal CA field (CA2) and blood vessel (BV). Magnifications are × 400. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. sham-injured mice; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. placebo mice; + +p < 0.01, and +++p < 0.001 vs. Gsn-L mice by ANOVA, Newman-Keuls post-hoc test. Data are means ± SD for n = 6-8.

Article Snippet: Sections used for immunocytochemistry were incubated in 0.3% hydrogen peroxide (H2O2) for 10 min, and incubated free-floating in antibodies (Abs) of polyclonal anti-mouse ionized calcium-binding adapter molecule 1 (Iba-1, 1:1000; Wako, Osaka, Japan), monoclonal anti-mouse CD11b (Mac-1, 1:1000; EuroBioScience, Lund, Sweden), monoclonal anti-mouse CD45 (1:1000; EuroBioScience), or rabbit anti-cleaved caspase-3 (1:50; Cell Signaling, Danvers, MA, USA) with 3% normal goat serum, 0.05%Triton-X in PBS, for 24-48 h rotating at 4°C.

Techniques: Migration, Derivative Assay