anti cd45 2 apc cy7  (Miltenyi Biotec)

Journal: Cell Death & Disease

Article Title: RIPK1/RIPK3 promotes vascular permeability to allow tumor cell extravasation independent of its necroptotic function

doi: 10.1038/cddis.2017.20

Figure Lengend Snippet: Loss of RIPK3 in the microenvironment does not alter early immune and cytokine response upon B16-F10 injection and bone marrow reconstitution shows the stromal compartment to be responsible. ( a ) B16-F10 luciferase cells home to the lung in both Wt and Ripk3 −/− mice measured by IVIS luminescent imaging (representative shown, 4–5 mice per group, n =2). ( b ) Cytokine levels in total lung lysates were measured by multiplex bead assay and levels of CCL-2, CXCL-1, IL-1 β and TWEAK are shown ( n =2). ( c ) Corresponding immune cell infiltration analysis by flow cytometry shows inflammatory monocytes (SiglecF − Ly6G − Ly6C hi CCR2 + ) and neutrophil granulocyte (SiglecF − CD11b + Ly6G + ) populations. Populations were pre-gated on singlets, live cells, Ter119 − and CD45 + cells. ( d ) Tumor nodules were counted 14 dpi of B16-F10 into CD45.1 recipient mice reconstituted with CD45.2 Wt and CD45.2 Ripk3 −/− bone marrow cells ( n =3) and CD45.2 or Ripk3 −/− recipient mice reconstituted with CD45.1 or Ripk3 −/− bone marrow cells ( n =2). Each dot represents a mouse; statistical analysis done by one-way ANOVA and Bonferroni post-test. *** P <0.001

Article Snippet: The following antibodies were used: CD45.1-eFluor 450, CD45.2-APC-Cy7, CD45-brilliant violet 605, CD11b-PE-Cy7, Ly6C-brilliant violet 711, CD11c-brilliant violet 510, Ly6G-PerCP-Cy5.5, MHC-II-alexa fluor 700, CD103-FITC, SiglecF-brilliant violet 421, CD86-brilliant violet 650, Ter119-brilliant violet 421, CCR-2-APC (BioTechne), fixable live/dead eFluor 780 or PI for live/dead assays.

Techniques: Injection, Luciferase, Imaging, Multiplex Assay, Flow Cytometry

Journal: eLife

Article Title: Neutrophils promote CXCR3-dependent itch in the development of atopic dermatitis

doi: 10.7554/eLife.48448

Figure Lengend Snippet: ( A ) Exact permutation test (10,000 iterations, see Materials and methods) for significance of mean absolute log 2 fold change in gene expression at Day 8 (MC903 vs. ethanol) of custom-defined groups of genes for indicated categories (see ). ( B ) Log 2 fold change in gene expression (MC903 vs. ethanol) in mouse skin at indicated time points for key immune and mouse/human AD genes that were significantly differentially expressed for at least one time point in the MC903 model. Only genes from our initial list (see Materials and methods) differentially expressed at corrected p<0.05 and changing >2 fold between treatments for at least one condition are shown. Green bars = increased expression in MC903 relative to ethanol; magenta = decreased expression. Exact values and corrected p -values are reported in and Supplemental Data, respectively. D1 = 6 hr post-treatment; D2 = Day 2; D5 = Day 5; D8 = Day 8. ( C ) Scratching behavior of mice treated with MC903 or ethanol for indicated length of time (two-way ANOVA: **** p interaction <0.0001, F(2,409) = 13.25; Sidak’s multiple comparisons: p day 3 = 0.1309, n = 62,51 mice; * p day 5 = 0.0171, n = 69,56 mice; **** p day 8 < 0.0001, n = 92,85 mice). Exact values displayed in . ( D ) Log 2 fold change in gene expression of neutrophil chemoattractants (upper), Th2 cytokines (middle) and T cell chemoattractants (lower, from RNA-seq data). ( E ) Neutrophil counts in MC903- and ethanol-treated skin at indicated time points (two-way ANOVA: ** p treatment = 0.0023, F(1,102) = 9.82; Sidak’s multiple comparisons: p day 2 > 0.999, n = 4,4 mice; p day 3 = 0.9801, n = 5,5 mice; *** p day 5 = 0.0003, n = 6,8 mice; *** p day 8 = 0.0001, n = 40,38 mice). ( F ) Basophil counts in MC903- and ethanol-treated skin at indicated time points (two-way ANOVA: ** p treatment = 0.0051, F(1,102) = 8.17; Sidak’s multiple comparisons: p day 2 > 0.999, n = 4,4 mice; p day 3 = 0.8850, n = 5,5 mice; p day 5 = 0.0606, n = 6,8 mice; **** p day 8 < 0.0001, n = 40,38 mice). ( G ) CD4 + T cell counts in MC903- and ethanol-treated skin at indicated time points (two-way ANOVA: ** p time = 0.0042, F(1,44) = 9.10; p day 3 = 0.9998, n = 8,6 mice; p day 5 = 0.2223, n = 9,8 mice; ** p day 8 = 0.0021, n = 11,8 mice). Day 8 immune cell infiltrate represented as % of CD45 + cells in (see for all experimental conditions). Exact values displayed in and representative FACS plots for myeloid and T cell gating shown in and . For , data from mice receiving i.p. injection of PBS (see ) in addition to MC903 or EtOH are also included. ( H ) (Upper and Lower) Representative maximum intensity Z-projections from immunohistochemistry (IHC) of whole-mount mouse skin on Day 2 of the MC903 model. Skin was stained with neuronal marker beta-tubulin III (BTIII; green). Hair follicle autofluorescence is visible in the magenta channel. Images were acquired on a confocal using a 20x water objective. ( I ) Quantification of innervation (see Materials and methods) of mouse skin as determined from BTIII staining (*p=0.012; two-tailed t-test ( t = 3.114; df = 9); n = 7,4 images each from two mice per treatment). Day 1 IHC results as follows: 31.78 ± 18.39% (MC903) and 31.51 ± 16.43% (EtOH); p=0.988; two-tailed unpaired t-test; n = 6 images each from two mice per treatment. Exact values are reported in . ( J ) Quantification of CGRP + nerve fibers (see Materials and methods) in skin (**p=0.0083; two-tailed t-test ( t = 2.868; df = 25); n = 15, 12 images from three mice per treatment). Exact values are reported in . Representative images in . Figure 1—source data 1. Values displayed in the bar plot shown in . Figure 1—source data 2. Values displayed in the heat map shown in . Figure 1—source data 3. Values displayed in the bar plot shown in . Figure 1—source data 4. Values displayed in the bar plots shown in and . Figure 1—source data 5. Values displayed in the bar plots shown in and . Figure 1—source data 6. Values displayed in the heat map shown in . Figure 1—source data 7. Values displayed in the heat map shown in . Figure 1—source data 8. Values displayed in the heat map shown in . Figure 1—source data 9. Values displayed in the bar plot shown in .

Article Snippet: Antibody , CD45.2 Monoclonal Antibody (104), APC-Cy7, eBioscience; CD45.2-APC/Cy7 (104) (Mouse monoclonal, 1:200) , eBioscience , RRID: AB_1272175 ; Cat # 47-0454-82 , .

Techniques: Expressing, RNA Sequencing Assay, Injection, Immunohistochemistry, Staining, Marker, Two Tailed Test

Journal: eLife

Article Title: Neutrophils promote CXCR3-dependent itch in the development of atopic dermatitis

doi: 10.7554/eLife.48448

Figure Lengend Snippet: ( A ) Representative flow cytometry plots of cells collected from blood of mice injected with PBS or aGr1 (250 μg, i.p.) once-daily for five days concurrent with daily MC903 topical treatment. Shown are CD45.2 + CD11b + cells, plotted by Ly6G and Ly6C signal, with neutrophil (Neuts.) and inflammatory monocyte (IMs) populations indicated. Neutrophils were defined as Cd11b + Ly6G + Ly6C mid/high and IMs were defined as Cd11b + Ly6G - Ly6C high (see Materials and methods). ( B ) Representative flow cytometry plot as in A, depicting neutrophil and IM populations from blood collected on day 8. ( C ) (Left) Neutrophil counts in blood shown as % of Cd11b + cells from aGr1/MC903 (black triangles) and PBS/MC903 (gray circles)-treated animals on days 3, 5, and 8 of the model (two-way repeated measures ANOVA: **** p treatment <0.0001, F(1,31) = 299.5; Sidak’s multiple comparisons: **** p day 3 < 0.0001; **** p day 5 < 0.0001; **** p day 8 < 0.0001, n = 16,17 mice). (Right) Inflammatory monocyte counts in blood shown as % of Cd11b + cells from aGr1/MC903 and PBS/MC903-treated animals on days 3, 5, and 8 of the model (two-way repeated measures ANOVA: * p treatment = 0.0468, F(1,31) = 4.287; Sidak’s multiple comparisons: ** p day 3 = 0.0015; p day 5 = 0.1918; p day 8 = 0.2013, n = 16,17 mice). Exact values displayed in .

Article Snippet: Antibody , CD45.2 Monoclonal Antibody (104), APC-Cy7, eBioscience; CD45.2-APC/Cy7 (104) (Mouse monoclonal, 1:200) , eBioscience , RRID: AB_1272175 ; Cat # 47-0454-82 , .

Techniques: Flow Cytometry, Injection

Journal: eLife

Article Title: Neutrophils promote CXCR3-dependent itch in the development of atopic dermatitis

doi: 10.7554/eLife.48448

Figure Lengend Snippet: ( A ) Representative flow cytometry plots of cells from cheek skin of mice injected with PBS or CXCL1 (1 μg in 20 µL, s.c.). Shown are CD45.2 + CD11b + cells, plotted by Ly6G and Ly6C signal, with neutrophil and inflammatory monocyte (IMs) populations indicated. ( B ) Neutrophil count from cheek skin of mice 5, 15, and 30 min after injection of CXCL1 or PBS (two-way ANOVA: * p interaction = 0.0239, F(2,21) = 4.48; Sidak’s multiple comparisons: p 5 min >0.9999, n = 4,5 mice; * p day 15 min = 0.0141, n = 4,4 mice; ** p day 30 min = 0.0031, n = 3,7 mice). Exact values displayed in . ( C ) Blood neutrophils as % of Cd11b + cells approximately 20 hr after injection of 250 µg aGr1 (n = 15 mice). Mice assayed for CXCL1-evoked itch behavior immediately preceding blood isolation (see ). Exact values displayed in . See for representative blood neutrophil measurements from PBS-injected animals.

Article Snippet: Antibody , CD45.2 Monoclonal Antibody (104), APC-Cy7, eBioscience; CD45.2-APC/Cy7 (104) (Mouse monoclonal, 1:200) , eBioscience , RRID: AB_1272175 ; Cat # 47-0454-82 , .

Techniques: Flow Cytometry, Injection, Isolation

Journal: eLife

Article Title: Neutrophils promote CXCR3-dependent itch in the development of atopic dermatitis

doi: 10.7554/eLife.48448

Figure Lengend Snippet: ( A ) Exact permutation test (10,000 iterations, see Materials and methods) for significance of mean absolute log 2 fold change in gene expression at Day 8 (MC903 vs. ethanol) of custom-defined groups of genes for indicated categories (see ). ( B ) Log 2 fold change in gene expression (MC903 vs. ethanol) in mouse trigeminal ganglia (TG) at indicated time points for all genes which were significantly differentially expressed for at least one time point in the MC903 model. Green bars = increased expression in MC903 relative to ethanol; magenta = decreased expression. Exact values and corrected p -values are reported in and Supplemental Data, respectively. ( C ) Representative composite images showing immune cells (CD45, green), and sensory neurons (Prph, magenta) with DAPI (blue) in sectioned trigeminal ganglia from mice treated with Vehicle or MC903 for five days on the cheek. ( D ) Quantification of images examining average number of CD45 + cells per section and average ratio of CD45:Peripherin cells per section after five days of treatment (p=0.562 ( t = 0.6318, df = 4), 0.542 ( t = 0.6660, df = 4); two-tailed unpaired t-tests, n = 33–159 fields of view (images) each of both trigeminal ganglia from three mice per condition treated bilaterally). ( E ) Representative composite images showing immune cells (CD45, green), and sensory neurons (Peripherin (Prph), magenta) with DAPI (blue) in sectioned trigeminal ganglia from mice treated with Vehicle or MC903 for eight days on the cheek. ( F ) Quantification of images examining average number of CD45 + cells per section and average ratio of CD45:Peripherin cells per section after eight days of treatment (**p=0.0019 ( t = 5.977, df = 5), **p = 0.0093 ( t = 4.107, df = 4); two-tailed unpaired t-tests; n = 42–172 fields of view (images) each of both trigeminal ganglia from 3 EtOH or 4 MC903 animals treated bilaterally). Scale bar = 100 µm. Images were acquired on a fluorescence microscope using a 10x air objective. Values from bar plots and all TG IHC data are available in . ( G ) Log 2 fold change in gene expression (MC903 vs. ethanol) in mouse spinal cord on day 8 showing selected differentially expressed genes ( p adjusted < 0.05). Exact values and corrected p -values are reported in Supplemental Data. Figure 3—source data 1. Values displayed in the bar plot shown in . Figure 3—source data 2. Values displayed in the heat map shown in . Figure 3—source data 3. Quantification of all IHC samples from trigeminal ganglia, and Values displayed in the bar plots shown in .

Article Snippet: Antibody , CD45.2 Monoclonal Antibody (104), APC-Cy7, eBioscience; CD45.2-APC/Cy7 (104) (Mouse monoclonal, 1:200) , eBioscience , RRID: AB_1272175 ; Cat # 47-0454-82 , .

Techniques: Expressing, Two Tailed Test, Fluorescence, Microscopy

Journal: eLife

Article Title: Neutrophils promote CXCR3-dependent itch in the development of atopic dermatitis

doi: 10.7554/eLife.48448

Figure Lengend Snippet: ( A ) Representative composite image showing CD45 (green), Peripherin (magenta), and DAPI (blue). ( B ) Single-channel CD45 image with automated min/max intensity thresholding. ( C ) Resultant binary image generated from ( B ). ( D ) Cells were counted as the number of regions of interest (ROIs) outlined in blue.

Article Snippet: Antibody , CD45.2 Monoclonal Antibody (104), APC-Cy7, eBioscience; CD45.2-APC/Cy7 (104) (Mouse monoclonal, 1:200) , eBioscience , RRID: AB_1272175 ; Cat # 47-0454-82 , .

Techniques: Generated

Journal: eLife

Article Title: Neutrophils promote CXCR3-dependent itch in the development of atopic dermatitis

doi: 10.7554/eLife.48448

Figure Lengend Snippet:

Article Snippet: Antibody , CD45.2 Monoclonal Antibody (104), APC-Cy7, eBioscience; CD45.2-APC/Cy7 (104) (Mouse monoclonal, 1:200) , eBioscience , RRID: AB_1272175 ; Cat # 47-0454-82 , .

Techniques: Purification, Recombinant, Staining, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Titration, Protease Inhibitor, Flow Cytometry, Software