Journal: Diagnostic Pathology

Article Title: Establishment of monoclonal anti-human CD26 antibodies suitable for immunostaining of formalin-fixed tissue

doi: 10.1186/1746-1596-9-30

Figure Lengend Snippet: Flow cytometry analysis with novel anti-CD26 mAbs. A. Jurkat-CD26WT cells (red lines) or Jurkat parent cells (blue lines) were incubated with the hybridoma supernatant, and subsequently stained with PE-labeled anti-mouse Ig pAb, and analyzed by flow cytometry. B. Jurkat-CD26WT cells were incubated with the hybridoma supernatant (clone 1, 5, 11, 16, 18 or 19) or purified mouse anti-CD26 mAb (5F8) or commercial mouse anti-CD26 mAb (MBL, clone 44–4), and subsequently stained with PE-labeled anti-mouse Ig pAb, and analyzed by flow cytometry. The gray areas in each histogram show the data involving the isotype control. The mean fluorescence intensity (MFI) of each staining is shown. Data shown are repeated twice (A) and five times (B) with similar results.

Article Snippet: The sections were treated with 100 μl of hybridoma supernatants or purified mouse anti-human CD26 mAb or goat anti-human CD26 pAb for 2 hours at RT, and subsequently treated with HRP-conjugated horse anti-mouse Ig pAb or HRP-conjugated horse anti-goat IgG pAb (Vector Laboratories) for 30 min at RT.

Techniques: Flow Cytometry, Incubation, Staining, Labeling, Purification, Fluorescence

Journal: Diagnostic Pathology

Article Title: Establishment of monoclonal anti-human CD26 antibodies suitable for immunostaining of formalin-fixed tissue

doi: 10.1186/1746-1596-9-30

Figure Lengend Snippet: ELISA analysis with novel anti-CD26 mAbs. Non treated native soluble CD26 (sCD26) or urea treated denatured sCD26 was incubated with the hybridoma supernatant (clone 1, 5, 11, 16, 18 or 19) or purified mouse anti-CD26 mAb (5F8) or commercial mouse anti-CD26 mAb (MBL, clone 44–4) or purified goat anti-CD26 pAb (R&D Systems). The absorbance at 450 nm/570 nm was measured, and data are shown as mean ± S.E. from three independent experiments.

Article Snippet: The sections were treated with 100 μl of hybridoma supernatants or purified mouse anti-human CD26 mAb or goat anti-human CD26 pAb for 2 hours at RT, and subsequently treated with HRP-conjugated horse anti-mouse Ig pAb or HRP-conjugated horse anti-goat IgG pAb (Vector Laboratories) for 30 min at RT.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Purification

Journal: Diagnostic Pathology

Article Title: Establishment of monoclonal anti-human CD26 antibodies suitable for immunostaining of formalin-fixed tissue

doi: 10.1186/1746-1596-9-30

Figure Lengend Snippet: Representative results of immunostaining with novel anti-CD26 mAbs. A. The tissue specimens of liver, kidney, prostate or two cases of malignant mesothelioma were stained with 100 μl of commercial mouse anti-human CD26 mAb supernatant (MBL, clone 44–4) (i), or 10 μg/ml of purified goat anti-human CD26 pAb (R&D Systems) (ii), or newly developed hybridoma supernatant (clone 18 (iii), clone 19 (iv) or clone 3 (v)). B . Malignant mesothelioma tissue specimens were stained with commercial goat anti-human CD26 pAb (R&D Systems) (i), or purified novel mouse anti-human CD26 mAbs (clone 18 (ii) or clone 19 (iii)) at the indicated concentrations of Abs in the presence or absence of sCD26. C. The tissue specimens of hepatocellular carcinoma, renal cell carcinoma, prostate adenocarcinoma, colon adenocarcinoma or lung adenocarcinoma were stained with 100 μg/ml of commercial goat anti-human CD26 pAb (R&D Systems) (i), or purified mouse anti-human CD26 mAbs (clone 18 (ii) or clone 19 (iii)). All specimens were counterstained with hematoxylin (original magnification, 200X).

Article Snippet: The sections were treated with 100 μl of hybridoma supernatants or purified mouse anti-human CD26 mAb or goat anti-human CD26 pAb for 2 hours at RT, and subsequently treated with HRP-conjugated horse anti-mouse Ig pAb or HRP-conjugated horse anti-goat IgG pAb (Vector Laboratories) for 30 min at RT.

Techniques: Immunostaining, Staining, Purification

Journal: Diagnostic Pathology

Article Title: Establishment of monoclonal anti-human CD26 antibodies suitable for immunostaining of formalin-fixed tissue

doi: 10.1186/1746-1596-9-30

Figure Lengend Snippet: Analysis of crossreactivity of novel anti-CD26 mAbs with humanized anti-CD26 mAb. Jurkat-CD26WT cells were pretreated with unlabeled humanized anti-CD26 mAb (YS110) or human control IgG, and then treated with the hybridoma supernatant (clone 1, 5, 11, 16, 18 or 19) or purified mouse anti-CD26 mAb (1F7 or 5F8), and subsequently stained with PE-labeled anti-mouse Ig pAb. For staining with humanized anti-CD26 mAb, cells were stained with Alexa Fluor 647-labeled YS110 after pretreatment with unlabeled YS110. Data were analyzed by flow cytometry, and the percentage of mean fluorescence intensity (MFI) after YS110 blocking to MFI after control IgG blocking is shown. Data shown are repeated twice with similar results.

Article Snippet: The sections were treated with 100 μl of hybridoma supernatants or purified mouse anti-human CD26 mAb or goat anti-human CD26 pAb for 2 hours at RT, and subsequently treated with HRP-conjugated horse anti-mouse Ig pAb or HRP-conjugated horse anti-goat IgG pAb (Vector Laboratories) for 30 min at RT.

Techniques: Purification, Staining, Labeling, Flow Cytometry, Fluorescence, Blocking Assay

Journal: Diagnostic Pathology

Article Title: Establishment of monoclonal anti-human CD26 antibodies suitable for immunostaining of formalin-fixed tissue

doi: 10.1186/1746-1596-9-30

Figure Lengend Snippet: Blocking experiment of novel anti-CD26 mAb binding to CD26. Jurkat-CD26WT cells were pretreated with unlabeled mouse anti-CD26 mAbs (4G8, 1F7, 5F8, 16D4B, or 9C11) (blue lines) or mouse IgG 1 isotype control (Contl. IgG) (red lines), and subsequently stained with PE-labeled anti-mouse Ig pAb (i) or Alexa Fluor 647-labeled anti-CD26 mAbs (clone 18 (ii) or clone 19 (iii) ), and analyzed by flow cytometry. The representative histograms of CD26 expression are shown, and the gray areas in each histogram show the data involving the isotype control. Data shown are repeated twice with similar results.

Article Snippet: The sections were treated with 100 μl of hybridoma supernatants or purified mouse anti-human CD26 mAb or goat anti-human CD26 pAb for 2 hours at RT, and subsequently treated with HRP-conjugated horse anti-mouse Ig pAb or HRP-conjugated horse anti-goat IgG pAb (Vector Laboratories) for 30 min at RT.

Techniques: Blocking Assay, Binding Assay, Staining, Labeling, Flow Cytometry, Expressing

Journal: Diagnostic Pathology

Article Title: Establishment of monoclonal anti-human CD26 antibodies suitable for immunostaining of formalin-fixed tissue

doi: 10.1186/1746-1596-9-30

Figure Lengend Snippet: Staining for CD26 expression on COS-7 cells transfected with CD26 deletion mutants by novel anti-CD26 mAbs. cDNA of deleted CD26 was cotransfected with GFP-expressing plasmid to COS-7 cells. After 24 h, the transfected cells were stained with Alexa Fluor 647-labeled anti-CD26 mAbs (YS110, clone 18 or clone 19) or isotype control, and analyzed by flow cytometry. Following gating for GFP positive cells among all acquired cells, the percentage of CD26 positive cells was analyzed. Data shown are repeated twice with similar results.

Article Snippet: The sections were treated with 100 μl of hybridoma supernatants or purified mouse anti-human CD26 mAb or goat anti-human CD26 pAb for 2 hours at RT, and subsequently treated with HRP-conjugated horse anti-mouse Ig pAb or HRP-conjugated horse anti-goat IgG pAb (Vector Laboratories) for 30 min at RT.

Techniques: Staining, Expressing, Transfection, Plasmid Preparation, Labeling, Flow Cytometry

Journal: Emerging Infectious Diseases

Article Title: Livestock Susceptibility to Infection with Middle East Respiratory Syndrome Coronavirus

doi: 10.3201/eid2302.161239

Figure Lengend Snippet: Presence of MERS-CoV receptor DPP4 (IHC) and of mucosubstances (PAS) in upper and lower respiratory tract tissues from sheep, pigs, llamas, and horses. A) In the nose, DPP4 (red cytoplasmic or membrane staining) was present on the lining epithelium of pigs, llamas, and horses but not sheep. PAS staining (magenta) demonstrated more mucous cells in the lining epithelium of sheep and horses and a layer of mucus on the lining epithelium of the horses. B) DPP4 (red cytoplasmic or membrane staining) was present on the lining epithelium of the trachea, bronchus/bronchioles, and alveoli in the pigs, llamas and horses but not in the sheep. Original magnification ×400 for all images. DPP4, dipeptidyl peptidase-4; IHC, immunohistochemistry; MERS-CoV, Middle East respiratory syndrome coronavirus; PAS, periodic acid–Schiff; term., terminal.

Article Snippet: In brief, we performed DPP4 IHC staining by using 5 μg/mL of polyclonal goat IgG anti-human DPP4 antibody (R&D Systems, Abingdon, UK) and peroxidase-labeled rabbit anti-goat IgG (1:200; DAKO; Agilent Technologies Company, Santa Clara, CA, USA) as a secondary antibody.

Techniques: Membrane, Staining, Immunohistochemistry

Journal: Journal of Pediatric Hematology/Oncology

Article Title: Chemotherapy-induced Alteration of SDF-1/CXCR4 Expression in Bone Marrow–derived Mesenchymal Stem Cells From Adolescents and Young Adults With Acute Lymphoblastic Leukemia

doi: 10.1097/mph.0000000000000220

Figure Lengend Snippet: FIGURE 3. Secretion and expression of SDF-1 in BM-MSCs. A, SDF-1 level was measured by enzyme-linked immunosorbent assay. (a) The bone marrow plasma level of SDF-1 in patients with ALL was significantly increased compared with that in healthy donors and ALL-c (*P < 0.05; n = 10). (b), The same difference was found when SDF-1 levels were measured in the supernatants of MSC cultures after incubation for 72 hours. B, Western blotting was performed to quantify total SDF-1 in the lysates of MSCs. SDF-1 expression in the MSCs of ALL was significantly higher than that in the MSCs of healthy donors and ALL-c. C, The difference was also found at the transcriptional level by real-time PCR (*P < 0.05, n = 10). D, Surface-CD26 expression on the MSCs from ALL patients measured by flow cytometry was significantly lower than that in healthy donors and ALL-c (*P < 0.05; n = 10). E, Total CD26 was measured by Western blot in the MSCs. The fold difference was shown among the 3 types of MSCs. F, The same difference was also found at the transcriptional level (*P < 0.05; n = 10). ALL indicates acute lymphoblastic leukemia; BM-MSC, bone marrow–derived mesenchymal stem cell; GADPH, glyceraldehyde- 3-phosphate dehydrogenase; PCR, polymerase chain reaction.

Article Snippet: Membranes were hybridized with the following primary antibodies: rabbit anti-human CXCR4 (1:1000; Abcam), goat antihuman SDF-1(1:500; R&D Systems), goat anti-human CD26 (1:1000; R&D Systems), and mouse antibody to glyceraldehyde-3-phosphate dehydrogenase (1:4000; Santa Cruz).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Incubation, Western Blot, Real-time Polymerase Chain Reaction, Flow Cytometry, Derivative Assay, Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Extracellular Adenine Nucleotide and Adenosine Metabolism in Calcific Aortic Valve Disease

doi: 10.1101/402685

Figure Lengend Snippet: Representative images of cultured primary endothelial and interstitial cells isolated from human non-stenotic aortic valves in the following days after isolation (A). Magnification 100x. The rates of ATP hydrolysis (B, C), AMP hydrolysis (D, E) and adenosine deamination (E, F) on the surface of human aortic valve endothelial cells (hVEC; B, D, F ) and interstitial cells (hVIC; C, E, G ) in the presence of specific ecto-enzyme inhibitors. Flow cytometry analysis (H-J) . Mean fluorescence intensity of cell-surface CD39, CD73 or CD26 (ADA-binding protein) for CD31 high positive endothelial cells (H) . Percentage of interstitial cells (Vim+) as myofibroblast like-interstitial cells (αSMA high /Sia-), myo-/osteoblast-like interstitial cells (αSMA int /Sia+) and osteoblast-like interstitial cells (αSMA-/Sia+) (I) and mean fluorescence intensity of cell-surface CD39, CD73 or CD26 (ADA-binding protein) for each type of interstitial cells (J) . Results are shown as mean ± SEM; n =9 (independent isolations from 3 patients), * p <0.05, *** p <0.001, **** p <0.0001 vs. without specific inhibitors ( B-G) or control staining (H, J) by one-way Anova followed by Holm-Sidak post hoc test (B-E, H-J) or student t -test ( F, G ).

Article Snippet: CD39 and CD73 were stained using mouse anti-human CD39 ( Novus ) and mouse anti-human CD73 ( Novus ) primary antibody, followed by a Cy3-conjugated goat anti-mouse secondary antibody ( JacksonImmuno ). eNPP1, ALP, ADA, vWF, CD26, CD45, A1R, A2aR, A2bR and A3R were stained using a rabbit anti-human eNPP1 ( Novus ), rabbit anti-human ALP ( Novus ), rabbit anti-human ADA ( Proteintech ), rabbit anti-human vWF ( Proteintech ), rabbit anti-human CD26 ( Genetex ), rabbit anti-human CD45 ( Genetex ), rabbit anti-human A1R ( Novus ), rabbit anti-human A2aR ( Novus ), rabbit anti-human A2bR ( Novus ), rabbit anti-human A3R primary antibody ( Novus ), followed by a Cy3-conjugated goat anti-rabbit secondary antibody ( JacksonImmuno ).

Techniques: Cell Culture, Isolation, Flow Cytometry, Fluorescence, Binding Assay, Staining

Journal: bioRxiv

Article Title: Extracellular Adenine Nucleotide and Adenosine Metabolism in Calcific Aortic Valve Disease

doi: 10.1101/402685

Figure Lengend Snippet: Flow cytometry analysis of CD45 positive cells (immune cells) as a percentage of total isolated cells after 1 st step of isolation (cells located in the upper layers of the valve) and 2 nd step of isolation (cells located in the deeper layers of the valve) (A) . The composition of stenotic aortic valve immune infiltrate (B) expressed as a percentage (%) of total CD45+ cells, including T helper cells (CD45+,CD4+), T cytotoxic cells (CD45+,CD8+), B cells (CD45+,CD19+), monocytes/macrophages (CD45+,CD11b+, CD14+) and granulocytes (CD45+,CD11b int , CD14-). Mean fluorescence intensity of cell-surface CD39, CD73 or CD26 (ADA-binding protein) for each type of isolated immune cells (C) . Results are shown as mean ± SEM; n =9 (independent isolations from 3 patients), * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001 vs. CD45- (A) , as indicated (B) or vs. control staining (C) by Student t -test (A) , one-way Anova followed by Tukey post hoc test (B), Holm-Sidak post hoc test (C) . The rates of ATP hydrolysis (D, E), AMP hydrolysis (F, G) and adenosine deamination (H, I) on the surface of human peripheral blood mononuclear cells (PBMC; D, F, H ) and human monocyte/macrophages SC; E, G, I ) in the presence of specific ecto-enzyme inhibitors. Results are shown as mean ± SEM; n =5-9, * p <0.05, *** p <0.001, **** p <0.0001 vs. without specific ecto-enzyme inhibitors (D-I) by one-way Anova followed by Holm-Sidak post hoc test (D-G) or Student t -test ( H, I ).

Article Snippet: CD39 and CD73 were stained using mouse anti-human CD39 ( Novus ) and mouse anti-human CD73 ( Novus ) primary antibody, followed by a Cy3-conjugated goat anti-mouse secondary antibody ( JacksonImmuno ). eNPP1, ALP, ADA, vWF, CD26, CD45, A1R, A2aR, A2bR and A3R were stained using a rabbit anti-human eNPP1 ( Novus ), rabbit anti-human ALP ( Novus ), rabbit anti-human ADA ( Proteintech ), rabbit anti-human vWF ( Proteintech ), rabbit anti-human CD26 ( Genetex ), rabbit anti-human CD45 ( Genetex ), rabbit anti-human A1R ( Novus ), rabbit anti-human A2aR ( Novus ), rabbit anti-human A2bR ( Novus ), rabbit anti-human A3R primary antibody ( Novus ), followed by a Cy3-conjugated goat anti-rabbit secondary antibody ( JacksonImmuno ).

Techniques: Flow Cytometry, Isolation, Fluorescence, Binding Assay, Staining