Journal: Cell reports

Article Title: m 6 A mRNA methylation-directed myeloid cell activation controls progression of NAFLD and obesity

doi: 10.1016/j.celrep.2021.109968

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Ly6G/C (141Pr) , Fluidigm , Cat#: 3141005B, Clone: RB6-8C5.

Techniques: Recombinant, SYBR Green Assay, Reporter Assay, Isolation, Gene Expression, Software

Journal: Gut

Article Title: Targeted depletion of an MDSC subset unmasks pancreatic ductal adenocarcinoma to adaptive immunity.

doi: 10.1136/gutjnl-2013-306271

Figure Lengend Snippet: Figure 1 Cancer-conditioned myeloid cells chronicle the evolution of pancreatic ductal adenocarcinoma (PDA) in KPC mice. (A) The number of pancreatic Treg (CD45+CD4+FoxP3+), macrophages (CD45+CD11b+F4/80+), myeloid-derived suppressor cells (MDSC) (CD45+CD11b+RB6-8C5 [Ly6G/ Ly6C]+) and NK cells (CD45+NK1.1+) for each pancreas were quantified from normal pancreas (nl), 6–8-week-old KPC pancreata with confirmed preinvasive disease (Pre) and invasive tumours (PDA). Significant differences were detected in the number of Treg, tumour-associated macrophages and MDSC during disease progression. (B) Evolving profiles of three distinct populations of myeloid cells (gated on CD45+CD11b+) were seen in various organs based on expression patterns of Gr-1 and Ly6C. BM, bone marrow; LN, lymph node. (C) The percentages (top panel) and absolute numbers (bottom panel) of CD45+CD11b+ myeloid populations in the spleen and pancreas in normal (black filled circles) and preinvasive (grey filled circles) and invasive (open circles) disease settings. Data are plotted as mean±SEM and each data point represents an individual mouse. Granulocytic MDSC (Gr-MDSC)=CD45+CD11b+Gr-1highLy6Cint; monocytic MDSC (Mo-MDSC)=CD45+CD11b+Gr-1intLy6Chigh and macrophage (Mac) =CD45+CD11b+Gr1intLy6Cint. (D) Specific immunofluorescence reveals rare Ly6G/Ly6C+ (RB6-8C5) cells in normal pancreas, focal accumulation in pancreata with preinvasive disease and diffuse infiltration in invasive PDA. Specific Ly6G immunofluorescence demonstrates that Gr-MDSC are absent from normal pancreas, rare in preinvasive disease and abundant in invasive PDA. The majority of myeloid cells in normal pancreas and surrounding preinvasive lesions appear to be macrophages. Arrowheads, epithelial cells; arrows, myeloid cells; asterisk, Gr-MDSC. Scale bars, 50 mm. (E) Ly6G/Ly6C (RB6-8C5) staining in normal pancreas and KPC salivary gland. Arrowheads, epithelial cells; arrows, myeloid cells. Scale bars, 10 mm. *p<0.05; **p<0.005; ***p<0.0005.

Article Snippet: For immunofluorescence, OCT tissue sections (7 μm) were fixed in acetone at −20°C, blocked with phosphatebuffered saline (PBS)/1% bovine serum albumin (BSA) and incubated with the following primary antibodies: cleaved caspase-3 (Cell Signalling D175, 1:200), CD8α (BD Biosciences 53-6.7, 1:25), Gr-1 (eBioScience RB6-8C5, 1:50), Ly6G (Bioxcell 1A8, 1:50), PanCK-FITC (Sigma-Aldrich F3418, 1:200), SMA-1 (DAKO 1A4, 1:100), CD31 (BD Biosciences 390, 1:50), GMCSF (Biolegend, 1:50) or granzyme B (R&D Systems, 1:50).

Techniques: Derivative Assay, Biomarker Discovery, Expressing, Staining

Journal: Gut

Article Title: Targeted depletion of an MDSC subset unmasks pancreatic ductal adenocarcinoma to adaptive immunity.

doi: 10.1136/gutjnl-2013-306271

Figure Lengend Snippet: Figure 4 Systemic administration of 1A8 (αLy6G) specifically depletes Gr-myeloid-derived suppressor cells (Gr-MDSC) in autochthonous pancreatic ductal adenocarcinoma (PDA). (A) Representative myeloid cell profiles in peripheral blood mononuclear cells from a normal mouse and untreated (KPC) and 1A8-treated KPC (KPC + 1A8) mice. Numbers indicate the percentage of each subset gated on CD45 mononuclear cells. We note that the gates for the discrete subpopulations defined in the blood were then applied to the tissue-specific analyses. (B) Percentage of Gr-MDSC (squares) and monocytic MDSC (Mo-MDSC; circles) in blood after 1A8 treatment. Data represent mean±SD from three independently treated animals. (C) Representative fluorescence activated cell sorting (FACS) profiles of CD45+ CD11b+ splenocytes from control (−) and 1A8-treated (+) KPC mice (4–6 animals per group). (D) Both the percentage and number of splenic Gr-MDSC at endpoint of 1A8 treatment (day 12) are significantly decreased (**, p=0.005). (E) Representative FACS profiles of intratumoral myeloid cells in control (−) and 1A8-treated (+) KPC mice at day 12 of Gr-MDSC depletion. (F) The percentage and number of Gr-MDSC in PDA at the endpoint are significantly decreased in 1A8-treated mice compared with control KPC mice.

Article Snippet: For immunofluorescence, OCT tissue sections (7 μm) were fixed in acetone at −20°C, blocked with phosphatebuffered saline (PBS)/1% bovine serum albumin (BSA) and incubated with the following primary antibodies: cleaved caspase-3 (Cell Signalling D175, 1:200), CD8α (BD Biosciences 53-6.7, 1:25), Gr-1 (eBioScience RB6-8C5, 1:50), Ly6G (Bioxcell 1A8, 1:50), PanCK-FITC (Sigma-Aldrich F3418, 1:200), SMA-1 (DAKO 1A4, 1:100), CD31 (BD Biosciences 390, 1:50), GMCSF (Biolegend, 1:50) or granzyme B (R&D Systems, 1:50).

Techniques: Derivative Assay, FACS, Control

Journal: Science Advances

Article Title: Inflammation-triggered self-immolative conjugates enable oral peptide delivery by overcoming gastrointestinal barriers

doi: 10.1126/sciadv.aea2989

Figure Lengend Snippet: ( A ) Representative trajectories of Cy5-proKPV NPs and Cy5-labeled PS NPs in the mouse intestinal mucus, as determined by MTrackJ analysis of 5-min confocal microscopy recordings. ( B ) Time-dependent ensemble-averaged geometric MSD of proKPV NPs and PS NPs in the mouse intestinal mucus. ( C ) Distributions of log( D eff ) for proKPV NPs and PS NPs at a 5-min timescale, with at least 100 particles tracked per sample for each type. ( D ) Three-dimensional fluorescence images illustrate the penetration of Cy5-KPV and Cy5-proKPV NPs through the mouse intestinal mucus. ( E ) Representative ex vivo images (left) and quantitative analysis (right) illustrating accumulation of Cy5-labeled proKPV in the colons of mice with or without DSS-induced acute colitis at 6 hours after oral administration. ( F and G ) Fluorescence images (F) and quantitative analysis (G) of the colonic tissues isolated from colitis mice at different time points following treatment with free Cy5-KPV or Cy5-proKPV. ( H ) AUC of Cy5 fluorescence in colonic tissues after treatment with free Cy5-KPV or Cy5-proKPV. ( I ) Fluorescence images of cryosections of colonic tissues from colitis mice at 6 hours after treatment with Cy5-KPV or Cy5-proKPV. Nuclei were stained with DAPI (blue). ( J to L ) Immunofluorescence analysis of colocalization of Cy5-KPV or Cy5-proKPV with CK18 + intestinal epithelial cells (J), CD68 + macrophages (K), or Ly6G + neutrophils (L) in cryosections of colonic tissues. Data in (E), (G), and (H) are presented as means ± SD ( n = 3 biological replicates). * P < 0.05; *** P < 0.001.

Article Snippet: The obtained cryosections were separately stained with anti–cytokeratin 18 (CK18) antibody (bsm-52058R, Bioss Biotechnology, China), anti-F4/80 antibody (30325, Cell Signaling Technology, USA), or anti-Ly6G antibody (31469, Cell Signaling Technology, USA) to further evaluate the tissue distribution of NPs.

Techniques: Labeling, Confocal Microscopy, Fluorescence, Ex Vivo, Isolation, Staining, Immunofluorescence

Journal: Science Advances

Article Title: Inflammation-triggered self-immolative conjugates enable oral peptide delivery by overcoming gastrointestinal barriers

doi: 10.1126/sciadv.aea2989

Figure Lengend Snippet: ( A ) Schematic illustration of treatment regimens. ( B ) Body weight of mice during a 7-day treatment course. Data were normalized as the percentages of the body weight at day 0.** P < 0.01; *** P < 0.001; n.s., not significant versus the colitis group. ( C ) Changes in DAI. ** P < 0.01 and *** P < 0.001 versus the colitis group. ( D ) Digital photos (left) and quantified lengths (right) of colonic tissues isolated from mice at day 7 following different treatments. Scale bars, 5 mm. ( E ) Representative miniendoscopic images of colons from mice after 7 days of treatment. ( F ) Colonic tissue sections stained with H&E or PAS for different groups. ( G ) Immunofluorescence analysis of the expression of CK18, occludin, and ZO-1 in colonic tissues from mice following various treatments. Scale bars, 100 μm. ( H to M ) Levels of TNF-α (H), IL-1β (I), IL-6 (J), MPO (K), MDA (L), and ROS (M) in colonic tissues isolated from healthy or diseased mice treated with different formulations. After 7 days of treatment, colonic tissue homogenates were prepared, and mediator concentrations were quantified and normalized to the total protein content. ( N ) Immunofluorescence of colonic tissues showing Ly6G + neutrophils, CD206 + M2 macrophages, CitH3/NE-positive NETs, and FOXP3 + T reg cells after different treatments. Data in (B) to (D) and (H) to (M) are presented as means ± SD ( n = 6 biological replicates). * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant.

Article Snippet: The obtained cryosections were separately stained with anti–cytokeratin 18 (CK18) antibody (bsm-52058R, Bioss Biotechnology, China), anti-F4/80 antibody (30325, Cell Signaling Technology, USA), or anti-Ly6G antibody (31469, Cell Signaling Technology, USA) to further evaluate the tissue distribution of NPs.

Techniques: Isolation, Staining, Immunofluorescence, Expressing

Journal: Science Advances

Article Title: Inflammation-triggered self-immolative conjugates enable oral peptide delivery by overcoming gastrointestinal barriers

doi: 10.1126/sciadv.aea2989

Figure Lengend Snippet: ( A ) Body weight changes of mice during 7-day treatment, which were normalized to the baseline at day 0. ** P < 0.01 and *** P < 0.001; n.s., not significant versus the colitis group. ( B ) Changes in DAI values. ** P < 0.01 and *** P < 0.001 versus the colitis group. ( C and D ) Digital photos (C) and quantified lengths (D) of colonic tissues isolated from mice after 7 days of treatment. ( E ) Histological sections of colonic tissues stained with H&E or PAS. ( F ) Immunofluorescence indicates expression patterns of CK18, occludin, and ZO-1 in colonic tissues. ( G to J ) Levels of TNF-α (G), IL-6 (H), IL-1β (I), and MPO (J) in colonic tissues isolated from healthy or diseased mice treated with different formulations. ( K and L ) Immunofluorescence analysis of NETs (K), Ly6G + neutrophils, and CD206 + macrophages (L) in colonic tissues. Data in (A), (B), (D), and (G) to (J) are presented as means ± SD ( n = 6 biological replicates). * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant.

Article Snippet: The obtained cryosections were separately stained with anti–cytokeratin 18 (CK18) antibody (bsm-52058R, Bioss Biotechnology, China), anti-F4/80 antibody (30325, Cell Signaling Technology, USA), or anti-Ly6G antibody (31469, Cell Signaling Technology, USA) to further evaluate the tissue distribution of NPs.

Techniques: Isolation, Staining, Immunofluorescence, Expressing

Journal: Oncotarget

Article Title: Oncogenic KIT-induced aggressive systemic mastocytosis requires SHP2/PTPN11 phosphatase for disease progression in mice

doi:

Figure Lengend Snippet: A. Representative images of spleens isolated from DBA/2 mice at day 13 following retro-orbital injection of saline (sham), or P815-NT cells, or P815-KD cells (5 × 10 3 in 100 μl; 3-4 mice/group; 3 experiments). B. Graph depicts spleen weights from saline-injected sham mice (n=3), or mice injected with P815-NT or P815–KD cells (n=11) at day 13 (mean ± SD; * indicates significant difference between groups, p <0.05). C. Splenocytes isolated from mice described above were analyzed by flow cytometry. Representative histograms are shown for splenocytes stained with FITC-Gr1/PE-CD11b/PEcy5-B220, with mean values shown for single and double positive populations (n=3; results are representative of 3 independent experiments). D. Graph depicts absolute cell numbers (x10 6 ) for Gr1 + , CD11b + , and B220 + cells in spleen from mice described above (* indicates significant difference between NT and KD, p <0.05). E. Representative histograms are shown for splenocytes stained with PE-KIT/PEcy5-CD45, with mastocytoma cells identified as double positives, from mice described above. F. Graph depicts the absolute mastocytoma cell numbers in spleen (mean ± SD; n=10; * indicates significant difference between NT and KD, p <0.05).

Article Snippet: [ ] Antibodies for flow cytometry included: PE-conjugated anti-mouse CD117 (2B8; BD Bioscience), PE/cy5-conjugated anti-mouse CD45 (eBioscience), PE conjugated anti-mouse CD11b (M1/70; Biolegend), PE/cy5-conjugated anti-mouse CD45R (B220) (RA3-6B2; Biolegend), FITC anti-mouse Gr1 (RB6-8C5; Cedarlane).

Techniques: Isolation, Injection, Saline, Flow Cytometry, Staining

Journal: Oncotarget

Article Title: Oncogenic KIT-induced aggressive systemic mastocytosis requires SHP2/PTPN11 phosphatase for disease progression in mice

doi:

Figure Lengend Snippet: A. BM cells were isolated from DBA/2 mice at day 13 following retro-orbital injection of saline (sham), or P815-NT cells, or P815-KD cells. BM cells were stained with FITC-Gr1/PE-CD11b/PEcy5-B220 and analyzed by flow cytometry. Graph depicts absolute cell numbers (x10 6 ) for Gr1 + , CD11b + , and B220 + cells in BM from sham (n=3), or mice injected with P815-NT or P815-KD cells (mean ± SD, n=11; * indicates significant difference between NT and KD, p <0.05). B. Representative histograms are shown for BM cells stained with PE-KIT/PEcy5-CD45, with mastocytoma cells identified as double positives, from mice described above. C. Graph depicts the absolute mastocytoma cell numbers in BM (mean ± SD; n=11; * indicates significant difference between NT and KD, p <0.05).

Article Snippet: [ ] Antibodies for flow cytometry included: PE-conjugated anti-mouse CD117 (2B8; BD Bioscience), PE/cy5-conjugated anti-mouse CD45 (eBioscience), PE conjugated anti-mouse CD11b (M1/70; Biolegend), PE/cy5-conjugated anti-mouse CD45R (B220) (RA3-6B2; Biolegend), FITC anti-mouse Gr1 (RB6-8C5; Cedarlane).

Techniques: Isolation, Injection, Saline, Staining, Flow Cytometry

Journal: Oncotarget

Article Title: Oncogenic KIT-induced aggressive systemic mastocytosis requires SHP2/PTPN11 phosphatase for disease progression in mice

doi:

Figure Lengend Snippet: A. Single cell suspensions from peripheral blood were isolated from DBA/2 mice at day 13 following retro-orbital injection of saline (sham), or P815-NT cells, or P815-KD cells. Following lysis of erythrocytes, cells were stained with FITC-Gr1/PE-CD11b/PEcy5-B220, and analyzed by flow cytometry. Graph depicts absolute cell numbers (x10 6 ) for Gr1 + , CD11b + , and B220 + cells in peripheral blood from sham (n=3), or mice injected with P815-NT or P815-KD cells (mean ± SD, n=9 from 3 separate experiments). B. Representative histograms are shown for peripheral blood cells stained with PE-KIT/PEcy5-CD45, with mastocytoma cells identified as double positives, from mice analyzed in 3 independent experiments as described above. C. Graph depicts the absolute mastocytoma cell numbers in peripheral blood (mean ± SD; n=9 from 3 separate experiments; * indicates significant difference between NT and KD, p <0.05).

Article Snippet: [ ] Antibodies for flow cytometry included: PE-conjugated anti-mouse CD117 (2B8; BD Bioscience), PE/cy5-conjugated anti-mouse CD45 (eBioscience), PE conjugated anti-mouse CD11b (M1/70; Biolegend), PE/cy5-conjugated anti-mouse CD45R (B220) (RA3-6B2; Biolegend), FITC anti-mouse Gr1 (RB6-8C5; Cedarlane).

Techniques: Isolation, Injection, Saline, Lysis, Staining, Flow Cytometry

Journal: STAR Protocols

Article Title: Murine brain tumor microenvironment immunophenotyping using mass cytometry

doi: 10.1016/j.xpro.2022.101357

Figure Lengend Snippet: Antibody master mix

Article Snippet: Anti-Mouse Ly-6G/C/Gr-1 (RB6-8C5)-174Yb—100 Tests , Fluidigm , SKU# 3174008B.

Techniques:

Journal: STAR Protocols

Article Title: Murine brain tumor microenvironment immunophenotyping using mass cytometry

doi: 10.1016/j.xpro.2022.101357

Figure Lengend Snippet:

Article Snippet: Anti-Mouse Ly-6G/C/Gr-1 (RB6-8C5)-174Yb—100 Tests , Fluidigm , SKU# 3174008B.

Techniques: Purification, Recombinant, Red Blood Cell Lysis, Centrifugation, Staining, Electron Microscopy, Antibody Labeling, Software, Sterility, Spectrophotometry