Journal: Cell metabolism

Article Title: Ejection of damaged mitochondria and their removal by macrophages ensure efficient thermogenesis in brown adipose tissue

doi: 10.1016/j.cmet.2022.02.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CD86 Antibody, anti-mouse, FITC, Miltenyi Biotec , Miltenyi Biotec , Cat# 130–123-672; RRID:AB_2889633.

Techniques: Immunofluorescence, Recombinant, Cytometry, Purification, shRNA, Cell Culture, Lysis, XF Assay, Staining, Isolation, Transfection, Live Cell Imaging, Mouse Assay, Software, Expressing

Journal: FEBS Open Bio

Article Title: Expression of nephronectin is inhibited by oncostatin M via both JAK/STAT and MAPK pathways

doi: 10.1016/j.fob.2015.04.001

Figure Lengend Snippet: Analysis of OSM signaling molecules. (A) RT-PCR analysis for presence of OSM receptor ( Osmr ) and gp130 in MC3T3-E1 cells. With the present primers, the size of the PCR products for Osmr and gp130 were 132 and 100 bp, respectively. (B) MC3T3-E1 cells were subjected to flow cytometry analysis for the presence of the OSM receptor (OSMR) on the cell surface. The black area shows MC3T3-E1 cells incubated with biotinylated anti-OSMR and FITC streptavidin. The gray area shows MC3T3-E1 cells incubated with biotinylated anti-rat IgG2b κ isotype and FITC streptavidin. All cells were found positive for the OSM receptor. (C) OSM-induced phosphorylation of STAT3 blocked by JAK inhibitor. MC3T3-E1 cells were pretreated with 10 μM of the JAK inhibitor for 1 h, then treated with 10 ng/ml of OSM for 15 min. Total cellular proteins were extracted, and used to determine levels of phosphorylated and total STAT3 . (D) Inhibition of down-regulation of OSM-induced Npnt expression by JAK inhibitor. MC3T3-E1 cells were treated with 10 ng/ml of OSM and 10 μM of the JAK inhibitor for 24 h. Total cellular RNA was extracted, and mRNA levels for Npnt and Gapdh were examined using real-time PCR analysis. Results are shown as the mean ± SD from 3 samples as compared to the level with 0 ng/ml of OSM and the JAK inhibitor. ∗∗ P < 0.01, Student’s t test relative to the level with 0 ng/ml of the JAK inhibitor.

Article Snippet: Next, the cells were incubated with biotinylated rat IgG2b κ isotype as a control (Becton, Dickinson and Company, Cat No. 553987) or a biotinylated anti-mouse OSM receptor (OSMR) antibody (Medical & Biological Laboratories CO., Ltd., Cat. No. D059-3), followed by incubation with FITC streptavidin (Medical & Biological Laboratories CO., Ltd., Cat. No. 554060).

Techniques: Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Incubation, Phospho-proteomics, Inhibition, Expressing, Real-time Polymerase Chain Reaction

Journal: American journal of respiratory cell and molecular biology

Article Title: Resident murine alveolar and peritoneal macrophages differ in adhesion of apoptotic thymocytes

doi: 10.1165/rcmb.2003-0255OC

Figure Lengend Snippet: CD11c/CD18 mediates binding of apoptotic thymocytes to AMø. Mø were pre-incubated with complete medium containing saturating concentration of mAb for 30 minutes, and then thymocytes were added in medium containing the same mAb concentration, and adhesion was assayed after an additional 15 minutes incubation. mAbs are M17/4 (anti-CD11a) (dark gray bars), M1/70 (anti-CD11b) (white bars), HL3 (anti-CD11c) (light gray bars) and GAME-46 (anti-CD18) (black bars). Data are expressed as a percentage of the result for the appropriate isotype control in the same experiment, and represent mean ± SEM of 6–9 wells in at least two experiments; *, p<0.05, compared to other mAbs in same panel, ANOVA with Tukey-Kramer post-hoc testing.

Article Snippet: Antibodies The following mAbs were purchased from PharMingen (San Diego, CA): M17/4 (anti-murine CD11a; rat IgG 2a , κ); M1/70 (anti-murine CD11b; rat IgG 2b , κ); HL3 (anti-murine CD11c; Armenian hamster IgG 1 , λ); C71/16 (anti-murine CD18; rat IgG 2a , κ); M18/2 (anti-murine CD18; rat IgG 2a , κ); GAME-46 (anti-murine CD18; rat IgG 1 , κ); Ha2/5 (anti-murine CD29; Armenian hamster IgM, κ); R1-2 (anti-murine CD49d; rat IgG 2b , κ); 5H10-27 (anti-murine CD49e; rat IgG 2b , κ); H9.2B8 (anti-murine CD51; Armenian hamster IgG 1 , λ); 2C9.G2 (anti-murine CD61; Armenian hamster IgG 1 , κ); R3-34 (control rat IgG 1 , κ); R35-95 (control rat IgG 2a , κ); A95-1 (control rat IgG 2b , κ) G235-2356 (control Armenian hamster IgG 1 , λ); A19-4 (control hamster IgG3. λ); A19-3 (control hamster IgG 1 , κ); G235-1(control hamster IgM). mAb 2F8 (rat IgG 2b ) against the murine scavenger receptor type I/II (CD204) was obtained from Serotec (Raleigh, NC).

Techniques: Binding Assay, Incubation, Concentration Assay

Journal: The Journal of Clinical Investigation

Article Title: Granulocyte-CSF links destructive inflammation and comorbidities in obstructive lung disease

doi: 10.1172/JCI98224

Figure Lengend Snippet: (A) Total BAL cells and differential BAL cell counts of the indicated 12-week-old mice; pooled data from n = 4–7 per group. C57 = C57BL/6, S–/– = SHIP-1–/–, G–/– = G-CSF–/–. (B) Images of cytospins from the indicated 12-week-old mice. Scale bar: 100 μm. Images are representative of n = 4–7 per group. (C) Flow cytometry of CD11b versus CD11c expression on AMΦs from the indicated 12-week-old mice to segregate AMΦ subpopulations into subset R or Ri and Rii in SHIP-1–/– mice; representative of n = 10–12 per genotype in a minimum of 3 experiments. (D) Levels of chemokines KC and MIP-2 in the BALF of the 12-week-old indicated groups determined by multiplex assay; pooled data from n = 9–10 per group. (E) qRT-PCR analysis of G-CSFR gene expression in AMΦs from C57BL/6 mice (C57), CD11b– and CD11b+ AMΦs from SHIP-1–/– mice, sorted neutrophils (PMN) and bone marrow cells (BM) from 12-week-old mice; n = 2–6 per group pooled from 2 experiments. Assessment of (F) ERK and (G) STAT-3 phosphorylation by flow cytometry in AMΦs from C57BL/6 mice (C57) or SHIP-1–/– mice (S–/–) treated with media or 10 ng/ml G-CSF for 15 minutes. Data are expressed as a ratio to the unstimulated control; pooled data from n = 3–9 per group from 2 experiments. *P < 0.05; **P < 0.01; ****P < 0.0001 by ANOVA (A and D) or Mann-Whitney U test (F and G).

Article Snippet: Single-cell suspensions from BAL or spleen were prepared as described previously ( 82 ) and analyzed by flow cytometry on an LSRII flow cytometer (BD Biosciences) using the following monoclonal antibodies: FITC-anti-CD45 (rat IgG 2b , κ, 1:500, catalog 553080; BD Biosciences), PE-Cy-7-anti-CD11c (Armenian hamster, IgG, 1:500, catalog 25-0114-82; eBioscience), APC-e780-anti-CD11b (rat IgG 2b , κ, 1:500, catalog 47-0112-82; eBioscience), biotin-anti-Ly6C (rat IgM, κ, 1:500, catalog 557359; BD Biosciences), PE-anti-Ly6G (rat IgG 2a , κ, 1:500, catalog 551461; BD Biosciences), biotin-anti–c-fms (rat IgG 2a , κ, 1:500, catalog 13-1152-82; eBioscience), Ter119 (rat IgG 2b , κ, 1:500, catalog 553673; BD Biosciences), FITC-anti-CD71 (rat IgG 1 , κ, 1:500, catalog 553266; BD Biosciences), APC-e780-B220 (rat IgG 2a , κ, 1:500, catalog 47-0452-82; eBioscience), and eFluor 450 anti–MHC Class II (I-A/I-E) (rat IgG 2b , κ, 1:500, catalog 48-5321-82; eBioscience).

Techniques: Flow Cytometry, Expressing, Multiplex Assay, Quantitative RT-PCR, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Granulocyte-CSF links destructive inflammation and comorbidities in obstructive lung disease

doi: 10.1172/JCI98224

Figure Lengend Snippet: (A) Images of cytospins from the indicated 12-week-old mice at the indicated days after transnasal LPS challenge. Scale bar: 100 μm. Images are representative of n = 2–6 mice per group collected over 4 experiments. (B) Frequency of neutrophils (CD45intLy6G+CD11b+) and monocytes (CD45+Ly6G–CD11b+) in BALF at the indicated time points after LPS challenge measured by flow cytometry; pooled data from n = 2–6 per group collected over 4 experiments. Graphical data represent mean ± SEM for each group at each time point. C57 = C57BL/6, S–/– = SHIP-1–/–, G–/– = G-CSF–/–.

Article Snippet: Single-cell suspensions from BAL or spleen were prepared as described previously ( 82 ) and analyzed by flow cytometry on an LSRII flow cytometer (BD Biosciences) using the following monoclonal antibodies: FITC-anti-CD45 (rat IgG 2b , κ, 1:500, catalog 553080; BD Biosciences), PE-Cy-7-anti-CD11c (Armenian hamster, IgG, 1:500, catalog 25-0114-82; eBioscience), APC-e780-anti-CD11b (rat IgG 2b , κ, 1:500, catalog 47-0112-82; eBioscience), biotin-anti-Ly6C (rat IgM, κ, 1:500, catalog 557359; BD Biosciences), PE-anti-Ly6G (rat IgG 2a , κ, 1:500, catalog 551461; BD Biosciences), biotin-anti–c-fms (rat IgG 2a , κ, 1:500, catalog 13-1152-82; eBioscience), Ter119 (rat IgG 2b , κ, 1:500, catalog 553673; BD Biosciences), FITC-anti-CD71 (rat IgG 1 , κ, 1:500, catalog 553266; BD Biosciences), APC-e780-B220 (rat IgG 2a , κ, 1:500, catalog 47-0452-82; eBioscience), and eFluor 450 anti–MHC Class II (I-A/I-E) (rat IgG 2b , κ, 1:500, catalog 48-5321-82; eBioscience).

Techniques: Flow Cytometry