Journal: Journal of translational medicine

Article Title: Cholesterol overload in macrophages drives metabolic dysfunction-associated steatohepatitis via inhibiting 7-dehydrocholesterol reductase in mice.

doi: 10.1186/s12967-024-05905-1

Figure Lengend Snippet: Fig. 1 Hepatic cholesterol accumulation and M1 macrophages infiltration in MASH. Six-week-old male C57BL/6J mice were fed either a NCD or a CDAHFD for six weeks (n = 3). (a) Body weight gain curves and (b) liver/body weight ratio was recorded. (c) Serum ALT and AST levels were detected. (d) Liver sections stained with H&E and ORO, and immunohistochemically for F4/80+ and CD11b+, 100× magnification. (e-f) Quantifies hepatic TC and TG levels. (g) NAS score. (h-j) Hepatic relative mRNA expression of Tnfa, Il1b and Il6. (k-l) Hepatic relative protein levels of CD86, ARG-1 and CD206. (m-o) Flow cytometry analysis of macrophages (live+CD45+F4/80+CD11b+) and M1 macrophages (live+CD45+F4/80+CD11b+iNOS+). (p-q) Flow cytometry analysis of TREM2 (live+CD45+F4/80+TREM2+). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 between groups

Article Snippet: Immunohistochemical analysis utilized anti-F4/80 (70076s) and anti-CD11b (17800s) antibodies from Cell Signaling Technology (CST, USA), and biotinylated goat anti-rabbit IgG (SA1022, BOSTER, China).

Techniques: Staining, Expressing, Flow Cytometry

Journal: Journal of translational medicine

Article Title: Cholesterol overload in macrophages drives metabolic dysfunction-associated steatohepatitis via inhibiting 7-dehydrocholesterol reductase in mice.

doi: 10.1186/s12967-024-05905-1

Figure Lengend Snippet: Fig. 3 Correlation between DHCR7 expression and macrophage polarization in MASH. (a) Relative expression of cholesterol biosynthesis and efflux genes in primary macrophages after ox-LDL (100 µg/ mL) or ox-LDL combined with statin treating for 24 h. (b) Expression levels of cholesterol biosyn thesis and efflux genes in primary macrophages polarized to M1 with LPS (100 ng/mL) or M2 with IL-4 (20 ng/mL) for 24 h. (c-d) Western blot analysis for DHCR7 protein in M1 and M2 macrophages, with densitometry. (e) Immunofluorescence staining for DHCR7 in the polarized-macrophages, 200× magnification. (f-g) Multiple immunofluorescence co-localization for DHCR7 and CD11B on livers of NCD or CDAHFD mice. (h-i) DHCR7 protein and mRNA levels in total liver and primary macrophages isolated from livers of mice on control and CDAHFD. (j-k) Immunofluorescence multi-labeling of liver sections from NC and MASH patients for DHCR7 and macrophage marker CD68 to determine the proportion of DHCR7+CD68+ cells among total CD68+ cells. Data are presented as mean ± SD, with n = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 between groups

Article Snippet: Immunohistochemical analysis utilized anti-F4/80 (70076s) and anti-CD11b (17800s) antibodies from Cell Signaling Technology (CST, USA), and biotinylated goat anti-rabbit IgG (SA1022, BOSTER, China).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Isolation, Control, Labeling, Marker

Journal: Journal of translational medicine

Article Title: Cholesterol overload in macrophages drives metabolic dysfunction-associated steatohepatitis via inhibiting 7-dehydrocholesterol reductase in mice.

doi: 10.1186/s12967-024-05905-1

Figure Lengend Snippet: Fig. 5 Myeloid-macrophage-specific DHCR7 deficiency exacerbates MASH progression. Wildtype (WT) and myeloid-specific DHCR7 knockout (DHCR7 cKO) mice were fed with a normal control diet (CON) or a CDAHFD for 10 weeks (n = 7). (a) Body weight progression, (b) liver/body weight ratio, and (c-d) serum ALT and AST levels were detected. (e-f) Hepatic TG and TC levels were measured. (g) ELISA quantification of liver IL-1β and IL-6 levels. (h) Histological analysis of liver sections is presented through H&E, ORO staining, and immunohistochemistry for F4/80+ and CD11b+ cells (200× magnification). (i) NAS score. (j-k) Relative protein levels of CD86, ARG-1, CD206 were determined, with densitometry. (l) Flow cytometry of primary macrophage in cKO mice and WT with different diets. (m) Flow cytometry of TREM2 in macrophages. (n) Predominantly of the M1 type in macrophages, including quantification of both the percentage of M1 macrophages and M1 macrophage counts per liver weight. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 between group

Article Snippet: Immunohistochemical analysis utilized anti-F4/80 (70076s) and anti-CD11b (17800s) antibodies from Cell Signaling Technology (CST, USA), and biotinylated goat anti-rabbit IgG (SA1022, BOSTER, China).

Techniques: Knock-Out, Control, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemistry, Flow Cytometry

Journal: Frontiers in Veterinary Science

Article Title: Flow cytometric-based detection of CD80 is a useful diagnostic marker of acute myeloid leukemia in dogs

doi: 10.3389/fvets.2024.1405297

Figure Lengend Snippet: Conjugated antibodies used for triple-labeling cells in acute leukemia immunophenotyping panels after April 2021, including their target antigen and registry number (when available) or source.

Article Snippet: CD11b-FITC*/CD22-PE or CD34-PE/CD11c-APC* , CD11b : Cell Signaling Technology Cat# 24442S, RRID: NA; CD22: Abcam Cat# ab23620, RRID:AB_447570, CD11c: BioLegend Cat# 117310 (also 117,309), RRID:AB_313779.

Techniques:

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Mesencephalic Astrocyte-derived Neurotrophic Factor Supports Hepatitis B Virus-induced Immunotolerance

doi: 10.1016/j.jcmgh.2024.05.008

Figure Lengend Snippet: MANF expression was increased in the liver of HBV-positive mice. C57BL/6 mice were sacrificed 3 weeks after the pAAV/HBV1.2 plasmid injection. ( A ) Relative mRNA expression of MANF in the liver of HBV-positive C57BL/6 mice was analyzed by q-PCR. ( B ) Immunofluorescence staining for CD11b ( green ), MANF ( red ), and DAPI for nuclei ( blue ) in HBV-positive WT mice liver tissues. Orange arrows point to CD11b + myeloid cells with high expression of MANF. ( C ) Immunofluorescence staining for Hepar1 ( green ), MANF ( red ), and DAPI for nuclei ( blue ) in HBV-positive WT mice liver tissues. Orange arrows point to high expression MANF in hepatocytes. Each experiment was repeated at least 3 times. Data were expressed as the mean ± standard error of the mean. n = 6 mice per group. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001.

Article Snippet: Frozen sections were stained with rabbit anti-mouse CD11b (1/50 dilution, #46512, Cell Signaling Technology), hepatocyte-specific antibody (HepPar1) (1/50 dilution, #NBP2-45272, Novus), and mouse anti-mouse MANF antibody, followed by Alexa Fluor 488 Conjugate anti-rat IgG (H + L) (1/1000 dilution, #4416, Cell Signaling Technology), Alexa Fluor 488 Conjugate anti-mouse IgG (H + L) (1/1000 dilution, #4408, Cell Signaling Technology), Rhodamine conjugated goat anti-mouse IgG (H + L) (1/100 dilution, ZSGB-BIO, ZF0313), and 4′, 6′-diamino-2-phenylindole (DAPI).

Techniques: Expressing, Plasmid Preparation, Injection, Immunofluorescence, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Mesencephalic Astrocyte-derived Neurotrophic Factor Supports Hepatitis B Virus-induced Immunotolerance

doi: 10.1016/j.jcmgh.2024.05.008

Figure Lengend Snippet: Myeloid MANF promotes HBV-induced tolerance via MDSCs. ( A ) The percentage and absolute number of MDSCs in liver tissues were analyzed by flow cytometry 3 weeks after pAAV/HBV1.2 plasmid injection. ( B–D ) The percentage and absolute number of PMN-MDSCs (CD11b + Ly6G + Ly6C low ) and M-MDSCs (CD11b + Ly6G - Ly6C high ) in liver tissues were analyzed by flow cytometry 3 weeks post-injection. ( E ) Schematic representation of the model of mice treatment. WT mice and Manf Mye-/- mice were injected with pAAV/HBV1.2 plasmid. Sorted MDSCs-derived from HBV-positive WT mice were transferred into Manf Mye-/- mice once a week. Mice were sacrificed on day 23 post-injection. ( F ) Serum HBsAg levels were assessed. ( G ) The percentage of hepatic IFN-γ + CD8 + T cells was examined. Each experiment was repeated twice. Data were expressed as the mean ± standard error of the mean. n = 5–7 mice per group. ∗ P < .05; ∗∗ P < .01.

Article Snippet: Frozen sections were stained with rabbit anti-mouse CD11b (1/50 dilution, #46512, Cell Signaling Technology), hepatocyte-specific antibody (HepPar1) (1/50 dilution, #NBP2-45272, Novus), and mouse anti-mouse MANF antibody, followed by Alexa Fluor 488 Conjugate anti-rat IgG (H + L) (1/1000 dilution, #4416, Cell Signaling Technology), Alexa Fluor 488 Conjugate anti-mouse IgG (H + L) (1/1000 dilution, #4408, Cell Signaling Technology), Rhodamine conjugated goat anti-mouse IgG (H + L) (1/100 dilution, ZSGB-BIO, ZF0313), and 4′, 6′-diamino-2-phenylindole (DAPI).

Techniques: Flow Cytometry, Plasmid Preparation, Injection, Derivative Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Mesencephalic Astrocyte-derived Neurotrophic Factor Supports Hepatitis B Virus-induced Immunotolerance

doi: 10.1016/j.jcmgh.2024.05.008

Figure Lengend Snippet: Myeloid MANF deficiency did not apparently affect the apoptosis and proliferation of MDSCs. ( A–D ) Manf Mye-/- mice and WT mice were injected with HBV1.2 plasmid via the tail vein and sacrificed after 3 weeks. ( A, B ) Flow cytometry analysis of CD45 + CD11b + Gr1 + cell death using dual 7AAD and Annexin 5 staining. Q2, late apoptotic cells; Q3, early apoptotic cells. ( C, D ) Percentages of Edu + cells out among CD45 + CD11b + Gr1 + cells in liver tissues and bone marrow of WT and Manf Mye-/- mice. Data are expressed as the mean ± standard error of the mean. n = 4–6 mice per group. ns, not significant.

Article Snippet: Frozen sections were stained with rabbit anti-mouse CD11b (1/50 dilution, #46512, Cell Signaling Technology), hepatocyte-specific antibody (HepPar1) (1/50 dilution, #NBP2-45272, Novus), and mouse anti-mouse MANF antibody, followed by Alexa Fluor 488 Conjugate anti-rat IgG (H + L) (1/1000 dilution, #4416, Cell Signaling Technology), Alexa Fluor 488 Conjugate anti-mouse IgG (H + L) (1/1000 dilution, #4408, Cell Signaling Technology), Rhodamine conjugated goat anti-mouse IgG (H + L) (1/100 dilution, ZSGB-BIO, ZF0313), and 4′, 6′-diamino-2-phenylindole (DAPI).

Techniques: Injection, Plasmid Preparation, Flow Cytometry, Staining

Journal: The EMBO Journal

Article Title: A G1‐like state allows HIV ‐1 to bypass SAMHD 1 restriction in macrophages

doi: 10.15252/embj.201696025

Figure Lengend Snippet: A–D Peritoneal macrophages from 6‐ to 10‐week‐old C57BL/6 mice were stained for the macrophage markers (A) F4/80, (B) CD11b, (C) MCM2 or (D) active DNA synthesis (EdU) 2 h after isolation. Scale bars: 20 μm. E–K Peritoneal macrophages were isolated from 6‐ to 10‐week‐old WT or SAMHD1 −/− (KO) mice. Cells were infected with VSV‐G‐pseudotyped HIV‐1 GFP for 48 h and analysed for (G) infection, (H) MCM2 expression, (I) EdU incorporation (EdU was added at the time of infection), and co‐localisation between infection and (J) MCM2 protein or (K) active DNA synthesis (by EdU incorporation). (E, F) Random microscopic fields and plot profiles show an example of immunofluorescence intensity along infected cells. Scale bars: 10 μm. On average, 10 4 cells in each experiment were recorded and analysed using Hermes WiScan cell‐imaging system and ImageJ ( n ≥ 2, mean ± s.e.m.; (ns) non‐significant; **P ‐value ≤ 0.01, unpaired t ‐test).

Article Snippet: Antibodies used were as follows: anti‐cdc2 (Cell Signaling Technology, Beverly, MA, USA); anti‐CDK2 (H‐298, Santa Cruz Biotechnology); anti‐pCDK2(Thr160) (Bioss Inc., MA, USA); anti‐CDK4 (DCS156, Cell Signaling Technology); anti‐CDK6 (B‐10, Santa Cruz Biotechnology); anti‐SAMHD1 (ab67820, Abcam, UK), beta‐actin (ab6276, abcam, UK); mouse anti‐MCM2 (BM‐28, BD Biosciences, UK); and rabbit anti‐MCM2 (SP85) from Sigma; pSAMHD1 (a kind gift from M. Benkirane) and ProSci (Poway, CA, USA); anti‐Geminin (NCL‐L‐Geminin, Leica); anti‐mouse F4/80 and CD11b (kind gift from S. Yona, UCL); anti‐human CD68, CD14, CD163, CD80, CD86, CD40 (kind gift from M. Noursadeghi).

Techniques: Staining, DNA Synthesis, Isolation, Infection, Expressing, Immunofluorescence, Imaging

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: CD11b is a Novel Alternate Receptor for CD154 during Alloimmunity

doi: 10.1111/ajt.15835

Figure Lengend Snippet: A, MACS-enriched CD11b+ cells (purity 60%) from CD40−/− mouse splenocytes were incubated with 20 μg /ml of mouse soluble CD154 or media alone as a control. Subsequently, cells were stained with anti-CD154, and anti-CD11b. The ability of sCD154 to bind to both CD11b+ cells (middle row) and CD11b− cells (bottom row) in the absence of CD40 was assessed. B, CD40−/− MACS-enriched CD11b+ cells were incubated with 20 μg /ml sCD154 in the presence of 50 μg of either the CD154/CD11b antagonist peptide cM7 (C-EQLKKSKTL-C), or scrambled control peptide scM7 (C-KLSLEKQTK-C) for 1h as described in Material and Methods. Surface CD154 was detected by flow cytometry. C-D, WT B6 recipients received BALB/c skin grafts plus 106 DST, and either no further treatment, anti-CD154, or anti-CD40 plus either the CD154/CD11b antagonist peptide cM7 (C-EQLKKSKTL-C), or scrambled control peptide scM7 (C-KLSLEKQTK-C) as described in Material and Methods. D. Graft survival was assessed and compared by log-rank test. n=10–15/group.

Article Snippet: Mice were treated with an i.p. injection of 250 μg CTLA4-Ig, anti-CD154 dAb ( 28 ), anti-CD40 (7-E1G2b) ( 33 ) (all kindly provided by Bristol-Myers Squibb, Princeton, NJ), anti-CD154 (clone MR-1), anti-CD11b (clone M1/70) (both from BioXcell) on days 0, 2, 4, and 6, and then once per week continuously until day 50 for skin graft survival experiments.

Techniques: Incubation, Staining, Flow Cytometry

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: CD11b is a Novel Alternate Receptor for CD154 during Alloimmunity

doi: 10.1111/ajt.15835

Figure Lengend Snippet: WT B6 recipients received BALB/c skin grafts plus 106 DST, and either no further treatment or anti-CD40 plus either the CD154/CD11b antagonist peptide cM7, or scM7 control peptide as described in Methods. Animals were sacrificed on day 10 and draining lymph nodes and skin grafts were analyzed. A, Representative flow cytometry plots summary data of absolute numbers of IFN-γ-producing CD8+ cells following ex vivo re-stimulation. B-D. Representative flow cytometry plots and summary data of absolute numbers of skin graft-infiltrating H-2Kb+ CD3+ CD8+ T cells (B), H-2Kb+ CD11b+cells (C) and H-2Kb+ F4/80+ cells (D). Data are representative of 3 independent experiments with a total 10–15 mice/group. *p<0.05 by one-way ANOVA with Kruskal-Wallis post-test. ns, not significant.

Article Snippet: Mice were treated with an i.p. injection of 250 μg CTLA4-Ig, anti-CD154 dAb ( 28 ), anti-CD40 (7-E1G2b) ( 33 ) (all kindly provided by Bristol-Myers Squibb, Princeton, NJ), anti-CD154 (clone MR-1), anti-CD11b (clone M1/70) (both from BioXcell) on days 0, 2, 4, and 6, and then once per week continuously until day 50 for skin graft survival experiments.

Techniques: Flow Cytometry, Ex Vivo

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: CD11b is a Novel Alternate Receptor for CD154 during Alloimmunity

doi: 10.1111/ajt.15835

Figure Lengend Snippet: WT B6 recipients received BALB/c skin grafts and 106 DST, and either no further treatment, anti-CD40 alone, or anti-CD40 plus anti-CD11b (clone M1/70) on days 0, 2, 4, 6 as described in Materials and Methods. Grafts were followed for survival. Data indicate that the addition of anti-CD11b significantly improved allograft survival relative to blockade of CD40 alone (B) (p<0.05). n=10–15/group.

Article Snippet: Mice were treated with an i.p. injection of 250 μg CTLA4-Ig, anti-CD154 dAb ( 28 ), anti-CD40 (7-E1G2b) ( 33 ) (all kindly provided by Bristol-Myers Squibb, Princeton, NJ), anti-CD154 (clone MR-1), anti-CD11b (clone M1/70) (both from BioXcell) on days 0, 2, 4, and 6, and then once per week continuously until day 50 for skin graft survival experiments.

Techniques: