isotype controls igg1 igg4  (BioLegend)

 
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    Name:
    PE Mouse IgG1 κ Isotype Ctrl ICFC
    Description:
    PE Mouse IgG1 κ Isotype Ctrl ICFC MOPC 21 Isotype Mouse IgG1 κ Reactivity Mouse Apps ICFC Size 25 tests
    Catalog Number:
    400139
    Price:
    50
    Applications:
    ICFC
    Conjugate:
    PE
    Size:
    25 tests
    Category:
    Isotype Control
    Preparation:
    The immunoglobulin was purified by affinity chromatography and conjugated with PE under optimal conditions The solution is free of unconjugated PE and unconjugated immunoglobulin
    Source:
    Mouse
    Quantity:
    1
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    Structured Review

    BioLegend isotype controls igg1 igg4
    PE Mouse IgG1 κ Isotype Ctrl ICFC MOPC 21 Isotype Mouse IgG1 κ Reactivity Mouse Apps ICFC Size 25 tests
    https://www.bioz.com/result/isotype controls igg1 igg4/product/BioLegend
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    isotype controls igg1 igg4 - by Bioz Stars, 2020-07
    94/100 stars

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    Related Articles

    Staining:

    Article Title: The Effect of Dexamethasone, Adrenergic and Cholinergic Receptor Agonists on Phospholipid Metabolism in Human Osteoarthritic Synoviocytes
    Article Snippet: .. After trypsinization, cells were stained with APC anti-human CD90 (clone 5E10) and PE anti-human CD45 (clone 2D1) or APC mouse IgG1 and PE mouse IgG1 antibodies (clone MOPC-21, BioLegend, San Diego, CA, USA). .. More than 80% of cells used in the experiments were stained positively for the fibroblast-specific antigen CD90 (87.1 ± 18.4%), whereas staining for CD45 was negative.

    Article Title: Inflammasome Assembly in the Chorioamniotic Membranes during Spontaneous Labor at Term
    Article Snippet: .. Choriodecidual leukocytes were then stained with anti-ASC-PE (clone HASC-71; catalog number 653903; BioLegend, San Diego, CA) or the IgG isotype control (catalog number 400139; BioLegend) for 30 min at 4°C in the dark. .. Finally, the stained cells were washed with 1 mL of 1× BD Perm/Wash Buffer, re-suspended in 0.5 mL of stain buffer, and acquired using the BD LSR Fortessa Flow Cytometer and BD FACSDiva 6.0 software.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Treg depletion in non-human primates using a novel diphtheria toxin-based anti-human CCR4 immunotoxin
    Article Snippet: .. APC‐anti‐human CD8 mAb (clone# RPA‐T8, cat# 301014), PE‐CD20 (clone# 2H7, cat302306), Biotin‐anti‐human CD16 mAb (clone# 3G8, cat# 302004), PE‐CD16 mAb (clone# 3G8, cat# 302008), FITC‐anti‐human CD14 mAb (clone# M5E2, cat# 301803), PerCp‐Cy5.5‐anti‐human CD11b (clone# M1/70, cat# 101228), PE‐anti‐human CD194 (CCR4) mAb (Clone# L291H4, cat# 359412), PE‐mouse IgG1, κ (clone# MOPC‐21, cat# 400139), Alexa Fluor® 647 anti‐human Foxp3 mAb (clone# 150D, cat# 320014), Alex Fluor 647 Mouse IgG1 κ (clone# MOPC‐21, cat# 400136), PE‐streptavidin (cat# 405204) and APC‐streptavidin (cat# 405207) were purchased from Biolegend. .. Human/rat CCR4 fluorescein mAb (clone 205410, cat# FAB1567F), human/rat CCR4 PE mAb (clone 205410, cat# FAB1567P) and mouse IgG2B fluorescein isotype control (clone# 133303, cat# IC0041F) were purchased from R & D Systems.

    Recombinase Polymerase Amplification:

    Article Title: Treg depletion in non-human primates using a novel diphtheria toxin-based anti-human CCR4 immunotoxin
    Article Snippet: .. APC‐anti‐human CD8 mAb (clone# RPA‐T8, cat# 301014), PE‐CD20 (clone# 2H7, cat#302306), Biotin‐anti‐human CD16 mAb (clone# 3G8, cat# 302004), PE‐CD16 mAb (clone# 3G8, cat# 302008), FITC‐anti‐human CD14 mAb (clone# M5E2, cat# 301803), PerCp‐Cy5.5‐anti‐human CD11b (clone# M1/70, cat# 101228), PE‐anti‐human CD194 (CCR4) mAb (Clone# L291H4, cat# 359412), PE‐mouse IgG1, κ (clone# MOPC‐21, cat# 400139), Alexa Fluor® 647 anti‐human Foxp3 mAb (clone# 150D, cat# 320014), Alex Fluor 647 Mouse IgG1 κ (clone# MOPC‐21, cat# 400136), PE‐streptavidin (cat# 405204) and APC‐streptavidin (cat# 405207) were purchased from Biolegend. .. Human/rat CCR4 fluorescein mAb (clone 205410, cat# FAB1567F), human/rat CCR4 PE mAb (clone 205410, cat# FAB1567P) and mouse IgG2B fluorescein isotype control (clone# 133303, cat# IC0041F) were purchased from R & D Systems.

    FACS:

    Article Title: Targeting aPKC disables oncogenic signaling by both the EGFR and the proinflammatory cytokine TNFα in glioblastoma
    Article Snippet: .. PE–mouse anti-human TNFα (#502908), PE–mouse IgG1k isotype control (#400139), anti-human CD11b–Pacific blue (#101235), anti-human CD64–Alexa Fluor 647 (#305012), anti-human CD23–APC–cyanine 7 (Cy7) (#338520), anti-human CD14-PECy7 (#325617), anti-human CD80–fluorescein isothiocyanate (#305205), and anti-human CD163 (#333608) antibodies used for FACS were obtained from BioLegend. .. Species-specific secondary antibodies for immunohistochemistry or immunofluorescence conjugated to Alexa 488 or 594 were obtained from Life Technologies.

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  • 85
    BioLegend igg2a azide free isotype control antibody
    DC-STAMP proteins are phosphorylated on tyrosine residues, interact with SHP-1 and CD16, and may be involved in signal transduction (A) Proteins were isolated from fresh human monocytes, immunoprecipitated (IP) with anti-DC-STAMP antibody 1A2 (lane 2) or with anti-CD16 antibody (lane 3). IP lysates were analyzed by SDS-PAGE followed by immunoblotting (IB) with 1A2. Arrow shows the DC-STAMP band (~53 kDa). (B) Proteins were isolated from fresh human monocytes, IP with anti-SHP1 antibody (lane 2) or with the control antibody mouse IgG (lane 3). IP lysates were analyzed by SDS-PAGE followed by IB with 1A2. Arrow shows the DC-STAMP band (~53 kDa). (C) Proteins were isolated from fresh human monocytes, IP with mouse <t>IgG2a</t> (lane 2), or anti-SHP1 antibody (lane3) or anti-DC-STAMP antibody 1A2 (lane 4). IP lysates were analyzed by SDS-PAGE followed by IB with anti-phosphotyrosine antibody 4G10. The bands of SHP-1 (~70 kDa) and DC-STAMP (~53 kDa) are marked by arrows. (D) Examination of 1A2 effects on DC-STAMP signaling. (I) Human monocytes were cultured in OC-promoting media (RANKL+M-CSF) in the absence (lanes 2 and 4) or presence (lanes 3 and 5) of 1A2 for 8 days. Proteins were isolated and IP with mouse IgG2a (lanes 2 and 3) or with anti-SHP-1 antibody (lanes 4 and 5), and subjected to SDS-PAGE, followed by IB with anti-phosphotyrosine antibody 4G10. Arrow indicates the location of the SHP-1 band. (II) Purified human monocytes were cultured in OC-promoting media (RANKL+M-CSF) in the absence (a to c) or the presence of 1A2 (d to f), or in the presence of mouse IgG2a isotype control (g to i). Cells were harvested and analyzed at 3 different time points (16hrs: a d; 40 hrs: b e; 64 hrs: c f) for phosphorylated PLC-γ2 expression by intracellular staining with the anti- PLC- γ2 antibody (pY759) and flow cytometric analysis.
    Igg2a Azide Free Isotype Control Antibody, supplied by BioLegend, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/igg2a azide free isotype control antibody/product/BioLegend
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    88
    BioLegend igg1 pe isotype control
    Immunohistochemical detection of CD20 + lymphocytes with intracellular staining for IL-17 . (A) Peripheral blood mononuclear cells were isolated from peripheral blood and stimulated with CytoStim followed by Brefeldin A and stained histochemically with Diff-Quick™. (B-I) Cells were then phenotyped for <t>IgG1-PE</t> isotype control (C), CD20-PE (E), or CD19-PE (H). The cells were then fixed and permeabilized and stained for IL-17-FITC, as seen in (C), (F), and (I), respectively. The cells were mounted on slides with antifade fluid supplemented with DAPI for nuclear staining (B, D, and G). Arrows in the middle column of panels depict triple-stained CD20 + (red) lymphocytes, which are IL-17 + (green). The right-hand column of panels depicts CD19 + lymphocytes (red), which are IL-17 - . Images were viewed at ×1,000 with an Olympus BX60 microscope with C-mount and a Nikon S10 digital camera. The size bar represents 10 μm unless otherwise stated. All specimens were processed at the same time, as described in Materials and methods. At least 10 fields of view were examined for each panel. DAPI, 4',6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; IL, interleukin; PE, phycoerythrin.
    Igg1 Pe Isotype Control, supplied by BioLegend, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/igg1 pe isotype control/product/BioLegend
    Average 88 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    igg1 pe isotype control - by Bioz Stars, 2020-07
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    91
    BioLegend igg1 isotype control antibody
    Mrp8/14 reduces rolling velocity of neutrophils in vitro and in vivo. Whole mouse blood was perfused through microflow chambers (shear stress 2.7 dyn cm −2 , n ≥3 mice per group). ( a ) Leukocytes rolling velocities of C57BL/6 WT mice were assessed in rmE-selectin ( n =209 cells) and rmE-selectin/rmICAM-1-coated glass capillaries ( n =178 cells). For rmE-selectin/rmICAM-1-coated glass capillaries, leukocyte rolling velocities were assessed for ( b ) untreated leukocytes (white bar, same bar as in a ), Paquinimod-treated leukocytes ( n =97 cells, grey spotted bar), rat <t>IgG2b</t> isotype-treated leukocytes ( n =165 cells, black bar), anti-TLR4 antibody 1A6-treated leukocytes ( n =184 cells, light grey bar) and Lovastatin-treated leukcoytes ( n =65 cells, grey lined bar). In addition, leukocyte rolling velocities from C57BL/6 WT mice were assessed for ( c ) untreated leukocytes (white bar, same bar as in a and b ), from Tlr4 −/− mice for untreated leukocytes ( n =271 cells, grey bar) or Paquinimod treated leukocytes ( n =182 cells, grey spotted bar) and from Rage −/− mice for untreated leukocytes ( n =88 cells, dark grey bar) or Paquinimod-treated leukocytes ( n =140 cells, dark grey spotted bar). ( d ) Ex vivo leukocyte rolling velocities of cells treated with control peptide ( n =140 cells, white bar) or MyD88 inhibitor IMG2005 ( n =160 cells, grey bar) were assessed. ( e ) In vivo leukocyte rolling velocities were analysed in rmTNF-α-stimulated venules of mouse cremaster muscles of C57BL/6 WT mice, pretreated with PBS/10% DMSO as control ( n =98 cells, white bar) or pretreated with Paquinimod ( n =71 cells, grey spotted bar). ( f ) In addition, in vivo rolling velocity was assessed for mice pretreated with rat IgG2b isotype control ( n =225 cells, white bar) or pretreated with rat anti-mouse TLR4 antibody 1A6 ( n =214 cells, light grey bar). Data are presented as mean±s.e.m. ( a , d , e , f ) unpaired t -test, ( b , c ) one-way analysis of variance with Dunnett's post-hoc test.
    Igg1 Isotype Control Antibody, supplied by BioLegend, used in various techniques. Bioz Stars score: 91/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/igg1 isotype control antibody/product/BioLegend
    Average 91 stars, based on 11 article reviews
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    90
    BioLegend rat isotype control igg
    CX3CL1 in EEV fractions promotes transmigration of prostate carcinoma cells. (a) Immunofluorescence of CD9 (red) and podoplanin (green) in the peritumoral stroma of a human prostate carcinoma (fluorescence profiles across respective vessels are plotted above the merged images; x axis, cross-sectional distance; y axis, CD9- or podoplanin-specific mean immunofluorescence intensities; n = 5). Bars, 10 µm. (b) Flow cytometry histogram of unstained (red), <t>APC-conjugated</t> isotype <t>IgG-stained</t> (blue), and APC-conjugated anti-CX3CR1 IgG-stained (green) PC3-ML prostate carcinoma cells. X axis, percentage of maximum; Y axis, mean fluorescence intensity (MFI) of APC-conjugated IgG ( n = 4). (c) Microfluidic adhesion setup (top). EEV fractions were immobilized in microfluidic capillaries, and the adhesions of PC3-ML prostate carcinoma cells were acquired under constant-flow conditions for 2 min. Brightfield images of cancer cells (bottom) that adhered to EEV-free supernatant-coated (left), ss-EEV fraction-coated (middle), or TNFα-EEV fraction-coated (right) microfluidic channels ( n = 3). Bars, 50 µm. (d) Automated quantitation of adherent PC3-ML cell tracks in EEV-free supernatant-coated, ss-EEV fraction-coated, or TNFα-EEV fraction-coated ( n = 3; unpaired two-tailed t test) microfluidic channels. (e) Brightfield images of transmigrated PC3-ML prostate carcinoma cells from the upper cell culture insert into the lower chamber well of a transwell assay. PC3-ML cells were loaded into the upper cell culture inserts after EEV-free supernatants (leftmost), ss-EEV fractions (center left), TNFα-EEV fractions (middle), TNFα-EEV fractions plus anti-CX3CL1 IgG (center right), or TNFα-EEV fractions plus isotype control IgG (rightmost) were loaded into the bottom chamber wells ( n = 3). Bars, 100 µm. (f) Quantitation of transmigrated PC3-ML cells described in d ( n = 3; unpaired two-tailed t test). ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01.
    Rat Isotype Control Igg, supplied by BioLegend, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    DC-STAMP proteins are phosphorylated on tyrosine residues, interact with SHP-1 and CD16, and may be involved in signal transduction (A) Proteins were isolated from fresh human monocytes, immunoprecipitated (IP) with anti-DC-STAMP antibody 1A2 (lane 2) or with anti-CD16 antibody (lane 3). IP lysates were analyzed by SDS-PAGE followed by immunoblotting (IB) with 1A2. Arrow shows the DC-STAMP band (~53 kDa). (B) Proteins were isolated from fresh human monocytes, IP with anti-SHP1 antibody (lane 2) or with the control antibody mouse IgG (lane 3). IP lysates were analyzed by SDS-PAGE followed by IB with 1A2. Arrow shows the DC-STAMP band (~53 kDa). (C) Proteins were isolated from fresh human monocytes, IP with mouse IgG2a (lane 2), or anti-SHP1 antibody (lane3) or anti-DC-STAMP antibody 1A2 (lane 4). IP lysates were analyzed by SDS-PAGE followed by IB with anti-phosphotyrosine antibody 4G10. The bands of SHP-1 (~70 kDa) and DC-STAMP (~53 kDa) are marked by arrows. (D) Examination of 1A2 effects on DC-STAMP signaling. (I) Human monocytes were cultured in OC-promoting media (RANKL+M-CSF) in the absence (lanes 2 and 4) or presence (lanes 3 and 5) of 1A2 for 8 days. Proteins were isolated and IP with mouse IgG2a (lanes 2 and 3) or with anti-SHP-1 antibody (lanes 4 and 5), and subjected to SDS-PAGE, followed by IB with anti-phosphotyrosine antibody 4G10. Arrow indicates the location of the SHP-1 band. (II) Purified human monocytes were cultured in OC-promoting media (RANKL+M-CSF) in the absence (a to c) or the presence of 1A2 (d to f), or in the presence of mouse IgG2a isotype control (g to i). Cells were harvested and analyzed at 3 different time points (16hrs: a d; 40 hrs: b e; 64 hrs: c f) for phosphorylated PLC-γ2 expression by intracellular staining with the anti- PLC- γ2 antibody (pY759) and flow cytometric analysis.

    Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

    Article Title: Regulation of Human Osteoclast Development by Dendritic Cell- Specific Tr ans membrane Protein (DC-STAMP)

    doi: 10.1002/jbmr.531

    Figure Lengend Snippet: DC-STAMP proteins are phosphorylated on tyrosine residues, interact with SHP-1 and CD16, and may be involved in signal transduction (A) Proteins were isolated from fresh human monocytes, immunoprecipitated (IP) with anti-DC-STAMP antibody 1A2 (lane 2) or with anti-CD16 antibody (lane 3). IP lysates were analyzed by SDS-PAGE followed by immunoblotting (IB) with 1A2. Arrow shows the DC-STAMP band (~53 kDa). (B) Proteins were isolated from fresh human monocytes, IP with anti-SHP1 antibody (lane 2) or with the control antibody mouse IgG (lane 3). IP lysates were analyzed by SDS-PAGE followed by IB with 1A2. Arrow shows the DC-STAMP band (~53 kDa). (C) Proteins were isolated from fresh human monocytes, IP with mouse IgG2a (lane 2), or anti-SHP1 antibody (lane3) or anti-DC-STAMP antibody 1A2 (lane 4). IP lysates were analyzed by SDS-PAGE followed by IB with anti-phosphotyrosine antibody 4G10. The bands of SHP-1 (~70 kDa) and DC-STAMP (~53 kDa) are marked by arrows. (D) Examination of 1A2 effects on DC-STAMP signaling. (I) Human monocytes were cultured in OC-promoting media (RANKL+M-CSF) in the absence (lanes 2 and 4) or presence (lanes 3 and 5) of 1A2 for 8 days. Proteins were isolated and IP with mouse IgG2a (lanes 2 and 3) or with anti-SHP-1 antibody (lanes 4 and 5), and subjected to SDS-PAGE, followed by IB with anti-phosphotyrosine antibody 4G10. Arrow indicates the location of the SHP-1 band. (II) Purified human monocytes were cultured in OC-promoting media (RANKL+M-CSF) in the absence (a to c) or the presence of 1A2 (d to f), or in the presence of mouse IgG2a isotype control (g to i). Cells were harvested and analyzed at 3 different time points (16hrs: a d; 40 hrs: b e; 64 hrs: c f) for phosphorylated PLC-γ2 expression by intracellular staining with the anti- PLC- γ2 antibody (pY759) and flow cytometric analysis.

    Article Snippet: We added an IgG2a-azide free isotype control antibody (BioLegend) to monocyte and PBMC cultures.

    Techniques: Transduction, Isolation, Immunoprecipitation, SDS Page, Cell Culture, Purification, Planar Chromatography, Expressing, Staining, Flow Cytometry

    Functional characterization of anti-DC-STAMP mAb 1A2 (A) A positive association was noted between DC-STAMP and CD16 expression. CD14+CD16+ monocytes demonstrated a higher surface expression of DC-STAMP than CD14+CD16-cells. Human PBMC were purified by Ficoll gradient and stained with an antibody cocktail composed of 7-AAD, anti-DC-STAMP and anti-CD16 antibodies. The expression of DC-STAMP on unstained, isotype control, CD14+CD16- and CD14+CD16+ cells are labeled in red, blue, orange and green, respectively. Commercially available anti-DC-STAMP polyclonal antibody KR104 was used for this analysis. (B) Lane 2: total proteins isolated from the RAW cell line; lane 3: proteins isolated from a RAW cell line expressing the PTHR-DC-STAMP fusion protein; lane 4: immunoprecipitated human monocyte proteins by 1A2; lane 5: immunoprecipitated human monocyte proteins by mouse IgG2a. All proteins were denatured, separated by 10% gradient protein gel, and probed with the anti-DC-STAMP mAb 1A2 on western blots. Pink and blue asterisks label the PTHR-DC-STAMP fusion protein and DC-STAMP native protein, respectively. (C) DC-STAMP expression on human PBMC and multinucleated giant cells from giant cell tumor of bone was detected by immunohistochemical (IHC) staining using 1A2, (a) (b). Human PBMC were purified by Ficoll gradient, embedded in paraffin for section, and stained with (a) mouse IgG2a isotype control, or (b) 1A2. (c) (d). Human biopsy samples collected from giant cell tumor were sectioned, and stained with (c) mouse IgG2a isotype control, or (d) 1A2. Both 1A2 and mouse IgG2a isotype control were diluted at 1:1500 for staining. The polarized expression of DC-STAMP in the multinucleated giant cells from a giant cell tumor was labeled by arrows. (D) The anti-DC-STAMP mAb 1A2 was able to block OC formation in vitro. Enriched human monocytes were cultured in the absence (a) or presence (b) of 1A2 for 8 days and TRAP-stained for visualization and enumeration of OC. (c) Enriched human monocytes and PBMC isolated from different subjects were cultured in the absence (open bar), presence of 1A2 (solid bar) or with IgG2a isotype control (slash bar) for 8 days. The concentrations of 1A2 used for monocytes and PBMC were 15 μg/ml and 150 μg/ml, respectively.

    Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

    Article Title: Regulation of Human Osteoclast Development by Dendritic Cell- Specific Tr ans membrane Protein (DC-STAMP)

    doi: 10.1002/jbmr.531

    Figure Lengend Snippet: Functional characterization of anti-DC-STAMP mAb 1A2 (A) A positive association was noted between DC-STAMP and CD16 expression. CD14+CD16+ monocytes demonstrated a higher surface expression of DC-STAMP than CD14+CD16-cells. Human PBMC were purified by Ficoll gradient and stained with an antibody cocktail composed of 7-AAD, anti-DC-STAMP and anti-CD16 antibodies. The expression of DC-STAMP on unstained, isotype control, CD14+CD16- and CD14+CD16+ cells are labeled in red, blue, orange and green, respectively. Commercially available anti-DC-STAMP polyclonal antibody KR104 was used for this analysis. (B) Lane 2: total proteins isolated from the RAW cell line; lane 3: proteins isolated from a RAW cell line expressing the PTHR-DC-STAMP fusion protein; lane 4: immunoprecipitated human monocyte proteins by 1A2; lane 5: immunoprecipitated human monocyte proteins by mouse IgG2a. All proteins were denatured, separated by 10% gradient protein gel, and probed with the anti-DC-STAMP mAb 1A2 on western blots. Pink and blue asterisks label the PTHR-DC-STAMP fusion protein and DC-STAMP native protein, respectively. (C) DC-STAMP expression on human PBMC and multinucleated giant cells from giant cell tumor of bone was detected by immunohistochemical (IHC) staining using 1A2, (a) (b). Human PBMC were purified by Ficoll gradient, embedded in paraffin for section, and stained with (a) mouse IgG2a isotype control, or (b) 1A2. (c) (d). Human biopsy samples collected from giant cell tumor were sectioned, and stained with (c) mouse IgG2a isotype control, or (d) 1A2. Both 1A2 and mouse IgG2a isotype control were diluted at 1:1500 for staining. The polarized expression of DC-STAMP in the multinucleated giant cells from a giant cell tumor was labeled by arrows. (D) The anti-DC-STAMP mAb 1A2 was able to block OC formation in vitro. Enriched human monocytes were cultured in the absence (a) or presence (b) of 1A2 for 8 days and TRAP-stained for visualization and enumeration of OC. (c) Enriched human monocytes and PBMC isolated from different subjects were cultured in the absence (open bar), presence of 1A2 (solid bar) or with IgG2a isotype control (slash bar) for 8 days. The concentrations of 1A2 used for monocytes and PBMC were 15 μg/ml and 150 μg/ml, respectively.

    Article Snippet: We added an IgG2a-azide free isotype control antibody (BioLegend) to monocyte and PBMC cultures.

    Techniques: Functional Assay, Expressing, Purification, Staining, Labeling, Isolation, Immunoprecipitation, Western Blot, Immunohistochemistry, Blocking Assay, In Vitro, Cell Culture

    Immunohistochemical detection of CD20 + lymphocytes with intracellular staining for IL-17 . (A) Peripheral blood mononuclear cells were isolated from peripheral blood and stimulated with CytoStim followed by Brefeldin A and stained histochemically with Diff-Quick™. (B-I) Cells were then phenotyped for IgG1-PE isotype control (C), CD20-PE (E), or CD19-PE (H). The cells were then fixed and permeabilized and stained for IL-17-FITC, as seen in (C), (F), and (I), respectively. The cells were mounted on slides with antifade fluid supplemented with DAPI for nuclear staining (B, D, and G). Arrows in the middle column of panels depict triple-stained CD20 + (red) lymphocytes, which are IL-17 + (green). The right-hand column of panels depicts CD19 + lymphocytes (red), which are IL-17 - . Images were viewed at ×1,000 with an Olympus BX60 microscope with C-mount and a Nikon S10 digital camera. The size bar represents 10 μm unless otherwise stated. All specimens were processed at the same time, as described in Materials and methods. At least 10 fields of view were examined for each panel. DAPI, 4',6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; IL, interleukin; PE, phycoerythrin.

    Journal: Arthritis Research & Therapy

    Article Title: Frequency of Th17 CD20+ cells in the peripheral blood of rheumatoid arthritis patients is higher compared to healthy subjects

    doi: 10.1186/ar3541

    Figure Lengend Snippet: Immunohistochemical detection of CD20 + lymphocytes with intracellular staining for IL-17 . (A) Peripheral blood mononuclear cells were isolated from peripheral blood and stimulated with CytoStim followed by Brefeldin A and stained histochemically with Diff-Quick™. (B-I) Cells were then phenotyped for IgG1-PE isotype control (C), CD20-PE (E), or CD19-PE (H). The cells were then fixed and permeabilized and stained for IL-17-FITC, as seen in (C), (F), and (I), respectively. The cells were mounted on slides with antifade fluid supplemented with DAPI for nuclear staining (B, D, and G). Arrows in the middle column of panels depict triple-stained CD20 + (red) lymphocytes, which are IL-17 + (green). The right-hand column of panels depicts CD19 + lymphocytes (red), which are IL-17 - . Images were viewed at ×1,000 with an Olympus BX60 microscope with C-mount and a Nikon S10 digital camera. The size bar represents 10 μm unless otherwise stated. All specimens were processed at the same time, as described in Materials and methods. At least 10 fields of view were examined for each panel. DAPI, 4',6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; IL, interleukin; PE, phycoerythrin.

    Article Snippet: The following morning, aliquots of 107 cells/mL were stimulated alone with 20 μL of CytoStim for 6 hours at 37°C, with the protein transport inhibitor Brefeldin A being added at a final concentration of 5 μg/mL after 2 hours of this incubation period., For phenotype analysis and intracellular staining of IL-17, aliquots of CytoStim-treated and untreated cells (106 PBMCs) were transferred to Eppendorf tubes and the cells were washed in cell staining buffer (BioLegend) and stained with mouse anti-human antibodies against CD20-PE, CD19-PE, or IgG1-PE isotype control and then fixed and permeabilized with fixation buffer (BioLegend) and permeabilization buffer (BioLegend), respectively, before being probed for IL-17 with rabbit anti-human IL-17-FITC or IgG1-FITC isotype control antibodies.

    Techniques: Immunohistochemistry, Staining, Isolation, Diff-Quik, Microscopy

    Gating and control rationale for CD20 + T-cell selection . (A) (i) Typical flow cytometry dot plot of the forward and side scatter of peripheral blood mononuclear cells (PBMCs). (ii) Dot plot of P1 gated cells (as indicated in A) stained with CD20-FITC and CD19-PE and shown in 'R2'. (iii) Dot plot of P1 gated cells, except 'R2', stained with CD20-FITC and CD3-CyQ. (B) (i) Typical flow cytometry dot plot of the forward and side scatter of PBMCs. (ii) Dot plot of P1 gated cells stained with isotype control IgG-FITC and isotype control IgG-PE and shown in 'R2' (iii). Dot plot of P1 gated cells, except 'R2', stained with isotype control IgG-FITC and isotype control CD3-CyQ. (C) (i) Typical flow cytometry dot plot of the forward and side scatter of PBMCs. (ii) Dot plot of P1 gated cells stained with CD20-FITC and CD19-PE post-RTX treatment and shown in 'R2'. (iii) Dot plot of P1 gated cells, except 'R2', stained with CD20-FITC and CD3-CyQ post-RTX incubation.

    Journal: Arthritis Research & Therapy

    Article Title: Frequency of Th17 CD20+ cells in the peripheral blood of rheumatoid arthritis patients is higher compared to healthy subjects

    doi: 10.1186/ar3541

    Figure Lengend Snippet: Gating and control rationale for CD20 + T-cell selection . (A) (i) Typical flow cytometry dot plot of the forward and side scatter of peripheral blood mononuclear cells (PBMCs). (ii) Dot plot of P1 gated cells (as indicated in A) stained with CD20-FITC and CD19-PE and shown in 'R2'. (iii) Dot plot of P1 gated cells, except 'R2', stained with CD20-FITC and CD3-CyQ. (B) (i) Typical flow cytometry dot plot of the forward and side scatter of PBMCs. (ii) Dot plot of P1 gated cells stained with isotype control IgG-FITC and isotype control IgG-PE and shown in 'R2' (iii). Dot plot of P1 gated cells, except 'R2', stained with isotype control IgG-FITC and isotype control CD3-CyQ. (C) (i) Typical flow cytometry dot plot of the forward and side scatter of PBMCs. (ii) Dot plot of P1 gated cells stained with CD20-FITC and CD19-PE post-RTX treatment and shown in 'R2'. (iii) Dot plot of P1 gated cells, except 'R2', stained with CD20-FITC and CD3-CyQ post-RTX incubation.

    Article Snippet: The following morning, aliquots of 107 cells/mL were stimulated alone with 20 μL of CytoStim for 6 hours at 37°C, with the protein transport inhibitor Brefeldin A being added at a final concentration of 5 μg/mL after 2 hours of this incubation period., For phenotype analysis and intracellular staining of IL-17, aliquots of CytoStim-treated and untreated cells (106 PBMCs) were transferred to Eppendorf tubes and the cells were washed in cell staining buffer (BioLegend) and stained with mouse anti-human antibodies against CD20-PE, CD19-PE, or IgG1-PE isotype control and then fixed and permeabilized with fixation buffer (BioLegend) and permeabilization buffer (BioLegend), respectively, before being probed for IL-17 with rabbit anti-human IL-17-FITC or IgG1-FITC isotype control antibodies.

    Techniques: Selection, Flow Cytometry, Cytometry, Staining, Incubation

    Mrp8/14 reduces rolling velocity of neutrophils in vitro and in vivo. Whole mouse blood was perfused through microflow chambers (shear stress 2.7 dyn cm −2 , n ≥3 mice per group). ( a ) Leukocytes rolling velocities of C57BL/6 WT mice were assessed in rmE-selectin ( n =209 cells) and rmE-selectin/rmICAM-1-coated glass capillaries ( n =178 cells). For rmE-selectin/rmICAM-1-coated glass capillaries, leukocyte rolling velocities were assessed for ( b ) untreated leukocytes (white bar, same bar as in a ), Paquinimod-treated leukocytes ( n =97 cells, grey spotted bar), rat IgG2b isotype-treated leukocytes ( n =165 cells, black bar), anti-TLR4 antibody 1A6-treated leukocytes ( n =184 cells, light grey bar) and Lovastatin-treated leukcoytes ( n =65 cells, grey lined bar). In addition, leukocyte rolling velocities from C57BL/6 WT mice were assessed for ( c ) untreated leukocytes (white bar, same bar as in a and b ), from Tlr4 −/− mice for untreated leukocytes ( n =271 cells, grey bar) or Paquinimod treated leukocytes ( n =182 cells, grey spotted bar) and from Rage −/− mice for untreated leukocytes ( n =88 cells, dark grey bar) or Paquinimod-treated leukocytes ( n =140 cells, dark grey spotted bar). ( d ) Ex vivo leukocyte rolling velocities of cells treated with control peptide ( n =140 cells, white bar) or MyD88 inhibitor IMG2005 ( n =160 cells, grey bar) were assessed. ( e ) In vivo leukocyte rolling velocities were analysed in rmTNF-α-stimulated venules of mouse cremaster muscles of C57BL/6 WT mice, pretreated with PBS/10% DMSO as control ( n =98 cells, white bar) or pretreated with Paquinimod ( n =71 cells, grey spotted bar). ( f ) In addition, in vivo rolling velocity was assessed for mice pretreated with rat IgG2b isotype control ( n =225 cells, white bar) or pretreated with rat anti-mouse TLR4 antibody 1A6 ( n =214 cells, light grey bar). Data are presented as mean±s.e.m. ( a , d , e , f ) unpaired t -test, ( b , c ) one-way analysis of variance with Dunnett's post-hoc test.

    Journal: Nature Communications

    Article Title: Extracellular MRP8/14 is a regulator of β2 integrin-dependent neutrophil slow rolling and adhesion

    doi: 10.1038/ncomms7915

    Figure Lengend Snippet: Mrp8/14 reduces rolling velocity of neutrophils in vitro and in vivo. Whole mouse blood was perfused through microflow chambers (shear stress 2.7 dyn cm −2 , n ≥3 mice per group). ( a ) Leukocytes rolling velocities of C57BL/6 WT mice were assessed in rmE-selectin ( n =209 cells) and rmE-selectin/rmICAM-1-coated glass capillaries ( n =178 cells). For rmE-selectin/rmICAM-1-coated glass capillaries, leukocyte rolling velocities were assessed for ( b ) untreated leukocytes (white bar, same bar as in a ), Paquinimod-treated leukocytes ( n =97 cells, grey spotted bar), rat IgG2b isotype-treated leukocytes ( n =165 cells, black bar), anti-TLR4 antibody 1A6-treated leukocytes ( n =184 cells, light grey bar) and Lovastatin-treated leukcoytes ( n =65 cells, grey lined bar). In addition, leukocyte rolling velocities from C57BL/6 WT mice were assessed for ( c ) untreated leukocytes (white bar, same bar as in a and b ), from Tlr4 −/− mice for untreated leukocytes ( n =271 cells, grey bar) or Paquinimod treated leukocytes ( n =182 cells, grey spotted bar) and from Rage −/− mice for untreated leukocytes ( n =88 cells, dark grey bar) or Paquinimod-treated leukocytes ( n =140 cells, dark grey spotted bar). ( d ) Ex vivo leukocyte rolling velocities of cells treated with control peptide ( n =140 cells, white bar) or MyD88 inhibitor IMG2005 ( n =160 cells, grey bar) were assessed. ( e ) In vivo leukocyte rolling velocities were analysed in rmTNF-α-stimulated venules of mouse cremaster muscles of C57BL/6 WT mice, pretreated with PBS/10% DMSO as control ( n =98 cells, white bar) or pretreated with Paquinimod ( n =71 cells, grey spotted bar). ( f ) In addition, in vivo rolling velocity was assessed for mice pretreated with rat IgG2b isotype control ( n =225 cells, white bar) or pretreated with rat anti-mouse TLR4 antibody 1A6 ( n =214 cells, light grey bar). Data are presented as mean±s.e.m. ( a , d , e , f ) unpaired t -test, ( b , c ) one-way analysis of variance with Dunnett's post-hoc test.

    Article Snippet: The complex was prepared essentially free of endotoxin contamination from human neutrophils as previously described . hMRP8/14 (5 μg ml−1 ), LPS (Sigma-Aldrich, 1 μg ml−1 ), PMA (Sigma-Aldrich, 1 μM) or PBS/0.1% BSA were added to vials containing mouse anti-human β2 integrin activation antibody KIM127 (Invivo, 10 μg ml−1 ) or mAB 24 (Hycult Biotech, 10 μg ml−1 ) or IgG1 Isotype control antibody (BioLegend, 10 μg ml−1 ) and warmed up to 37 °C for 5 min. Preincubated neutrophils and pre-warmed substance/antibody mix were pooled.

    Techniques: In Vitro, In Vivo, Mouse Assay, Ex Vivo

    Mrp8/14 increases leukocyte adhesion in TNF-α-stimulated cremaster muscle venules in vivo. ( a ) C57BL/6 WT mice were pretreated with carrier substance (PBS/10% DMSO, control)+PTx (white bar), a combination of Paquinimod+PTx (grey spotted bar), rat IgG2b isotype control+PTx (black bar) or a combination of rat anti-mouse TLR4 antibody 1A6+PTx (light grey bar). ( b ) One representative micrograph is shown. ( c ) C57BL/6 WT mice were pretreated with a combination of Paquinimod+PTx (grey spotted bar, same bar as in a ), a combination of 1A6+PTx (light grey bar, same bar as in a ), a combination of rat anti-mouse E-selectin Ab 9A9+Paquinimod+PTx (grey spotted bar), a combination of 9A9+1A6+PTx (light grey lined bar) or a combination of 1A6+Paquinimod+PTx (light grey spotted bar). Data are presented as mean±s.e.m. of at least three mice per group, one-way analysis of variance with Tukey's post-hoc test.

    Journal: Nature Communications

    Article Title: Extracellular MRP8/14 is a regulator of β2 integrin-dependent neutrophil slow rolling and adhesion

    doi: 10.1038/ncomms7915

    Figure Lengend Snippet: Mrp8/14 increases leukocyte adhesion in TNF-α-stimulated cremaster muscle venules in vivo. ( a ) C57BL/6 WT mice were pretreated with carrier substance (PBS/10% DMSO, control)+PTx (white bar), a combination of Paquinimod+PTx (grey spotted bar), rat IgG2b isotype control+PTx (black bar) or a combination of rat anti-mouse TLR4 antibody 1A6+PTx (light grey bar). ( b ) One representative micrograph is shown. ( c ) C57BL/6 WT mice were pretreated with a combination of Paquinimod+PTx (grey spotted bar, same bar as in a ), a combination of 1A6+PTx (light grey bar, same bar as in a ), a combination of rat anti-mouse E-selectin Ab 9A9+Paquinimod+PTx (grey spotted bar), a combination of 9A9+1A6+PTx (light grey lined bar) or a combination of 1A6+Paquinimod+PTx (light grey spotted bar). Data are presented as mean±s.e.m. of at least three mice per group, one-way analysis of variance with Tukey's post-hoc test.

    Article Snippet: The complex was prepared essentially free of endotoxin contamination from human neutrophils as previously described . hMRP8/14 (5 μg ml−1 ), LPS (Sigma-Aldrich, 1 μg ml−1 ), PMA (Sigma-Aldrich, 1 μM) or PBS/0.1% BSA were added to vials containing mouse anti-human β2 integrin activation antibody KIM127 (Invivo, 10 μg ml−1 ) or mAB 24 (Hycult Biotech, 10 μg ml−1 ) or IgG1 Isotype control antibody (BioLegend, 10 μg ml−1 ) and warmed up to 37 °C for 5 min. Preincubated neutrophils and pre-warmed substance/antibody mix were pooled.

    Techniques: In Vivo, Mouse Assay

    CX3CL1 in EEV fractions promotes transmigration of prostate carcinoma cells. (a) Immunofluorescence of CD9 (red) and podoplanin (green) in the peritumoral stroma of a human prostate carcinoma (fluorescence profiles across respective vessels are plotted above the merged images; x axis, cross-sectional distance; y axis, CD9- or podoplanin-specific mean immunofluorescence intensities; n = 5). Bars, 10 µm. (b) Flow cytometry histogram of unstained (red), APC-conjugated isotype IgG-stained (blue), and APC-conjugated anti-CX3CR1 IgG-stained (green) PC3-ML prostate carcinoma cells. X axis, percentage of maximum; Y axis, mean fluorescence intensity (MFI) of APC-conjugated IgG ( n = 4). (c) Microfluidic adhesion setup (top). EEV fractions were immobilized in microfluidic capillaries, and the adhesions of PC3-ML prostate carcinoma cells were acquired under constant-flow conditions for 2 min. Brightfield images of cancer cells (bottom) that adhered to EEV-free supernatant-coated (left), ss-EEV fraction-coated (middle), or TNFα-EEV fraction-coated (right) microfluidic channels ( n = 3). Bars, 50 µm. (d) Automated quantitation of adherent PC3-ML cell tracks in EEV-free supernatant-coated, ss-EEV fraction-coated, or TNFα-EEV fraction-coated ( n = 3; unpaired two-tailed t test) microfluidic channels. (e) Brightfield images of transmigrated PC3-ML prostate carcinoma cells from the upper cell culture insert into the lower chamber well of a transwell assay. PC3-ML cells were loaded into the upper cell culture inserts after EEV-free supernatants (leftmost), ss-EEV fractions (center left), TNFα-EEV fractions (middle), TNFα-EEV fractions plus anti-CX3CL1 IgG (center right), or TNFα-EEV fractions plus isotype control IgG (rightmost) were loaded into the bottom chamber wells ( n = 3). Bars, 100 µm. (f) Quantitation of transmigrated PC3-ML cells described in d ( n = 3; unpaired two-tailed t test). ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01.

    Journal: The Journal of Cell Biology

    Article Title: Lymphatic exosomes promote dendritic cell migration along guidance cues

    doi: 10.1083/jcb.201612051

    Figure Lengend Snippet: CX3CL1 in EEV fractions promotes transmigration of prostate carcinoma cells. (a) Immunofluorescence of CD9 (red) and podoplanin (green) in the peritumoral stroma of a human prostate carcinoma (fluorescence profiles across respective vessels are plotted above the merged images; x axis, cross-sectional distance; y axis, CD9- or podoplanin-specific mean immunofluorescence intensities; n = 5). Bars, 10 µm. (b) Flow cytometry histogram of unstained (red), APC-conjugated isotype IgG-stained (blue), and APC-conjugated anti-CX3CR1 IgG-stained (green) PC3-ML prostate carcinoma cells. X axis, percentage of maximum; Y axis, mean fluorescence intensity (MFI) of APC-conjugated IgG ( n = 4). (c) Microfluidic adhesion setup (top). EEV fractions were immobilized in microfluidic capillaries, and the adhesions of PC3-ML prostate carcinoma cells were acquired under constant-flow conditions for 2 min. Brightfield images of cancer cells (bottom) that adhered to EEV-free supernatant-coated (left), ss-EEV fraction-coated (middle), or TNFα-EEV fraction-coated (right) microfluidic channels ( n = 3). Bars, 50 µm. (d) Automated quantitation of adherent PC3-ML cell tracks in EEV-free supernatant-coated, ss-EEV fraction-coated, or TNFα-EEV fraction-coated ( n = 3; unpaired two-tailed t test) microfluidic channels. (e) Brightfield images of transmigrated PC3-ML prostate carcinoma cells from the upper cell culture insert into the lower chamber well of a transwell assay. PC3-ML cells were loaded into the upper cell culture inserts after EEV-free supernatants (leftmost), ss-EEV fractions (center left), TNFα-EEV fractions (middle), TNFα-EEV fractions plus anti-CX3CL1 IgG (center right), or TNFα-EEV fractions plus isotype control IgG (rightmost) were loaded into the bottom chamber wells ( n = 3). Bars, 100 µm. (f) Quantitation of transmigrated PC3-ML cells described in d ( n = 3; unpaired two-tailed t test). ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01.

    Article Snippet: 105 cells were stained with allophycocyanin (APC)-conjugated rat anti–human CX3CR1 IgG (BioLegend) or rat isotype control IgG (BioLegend) for 60 min at 4°C.

    Techniques: Transmigration Assay, Immunofluorescence, Fluorescence, Flow Cytometry, Cytometry, Staining, Quantitation Assay, Two Tailed Test, Cell Culture, Transwell Assay