Journal: Cell Reports

Article Title: Flow Cytometry of Mouse and Human Adipocytes for the Analysis of Browning and Cellular Heterogeneity

doi: 10.1016/j.celrep.2018.08.006

Figure Lengend Snippet:

Article Snippet: The human/mouse Anti-UCP1 antibody (MAB6158, monoclonal Mouse IgG 2B Clone # 536435, R&D Systems) was conjugated to Alexa647 (ThermoFisher Alexa Fluor 647 antibody labeling kit A20186) and incubated with adipocytes at 1:300 for 1 h. Mouse IgG2B Alexa Fluor 647-conjugated Isotype Control antibody (R&D systems) was used as a control.

Techniques: Control, Recombinant, Blocking Assay, Antibody Labeling, Software, Flow Cytometry

Journal: Diabetologia

Article Title: Phenotypically distinct anti-insulin B cells repopulate pancreatic islets after anti-CD20 treatment in NOD mice

doi: 10.1007/s00125-019-04974-y

Figure Lengend Snippet: Anti-insulin B cells repopulate pancreatic islets more rapidly than insulin-negative B cells after anti-CD20 treatment. Groups of VH125.hCD20/NOD mice, aged 6–8 weeks old, were injected with 2H7 anti-CD20 or IgG isotype control. Spleen, PLNs and pancreatic islets were analysed for insulin-positive and insulin-negative B cells at 8 weeks and 12 weeks post depletion by flow cytometry. ( a – f ) No. of cells from IgG control-treated (black circles) and 2H7-treated (grey squares) mice for insulin-negative B cells ( a – c ) and insulin-positive B cells ( d – f ) from spleen ( a , d ) PLNs ( b , e ) and islets ( c , f ). ( g – i ) Percentage of B cells repopulated at 8 and 12 weeks after treatment from spleen ( g ), PLNs ( h ) and islets ( i ) of mice shown in ( a – f ). Percentages were calculated as individual numbers from each 2H7-treated mouse / mean number from all IgG control antibody-treated mice. Horizontal lines represent the median value. Data represent three independent experiments. At 8 weeks, n = 7 (spleen), n = 7 (PLNs) and n = 9 (islets) for control IgG-treated mice and n = 10 (spleen), n = 9 (PLNs) and n = 12 (islets) for 2H7-treated mice. At 12 weeks, n = 8 (spleen), n = 6 (PLNs) and n = 7 (islets) for control IgG and n = 11 (spleen), n = 7 (PLNs) and n = 11 (islets) for 2H7-treated mice. * p < 0.05 (one-way ANOVA)

Article Snippet: Female VH125.hCD20/NOD mice, 6–8 weeks of age were chosen at random to receive either anti-hCD20 antibody (clone 2H7; Bio-XCell, West Lebanon, NH, USA) or control IgG2b antibody (clone MPC-11; Bio-XCell [ , , ]), as described previously [ ].

Techniques: Injection, Control, Flow Cytometry

Journal: Diabetologia

Article Title: Phenotypically distinct anti-insulin B cells repopulate pancreatic islets after anti-CD20 treatment in NOD mice

doi: 10.1007/s00125-019-04974-y

Figure Lengend Snippet: CD138 int anti-insulin B cells are enriched in pancreatic islets after anti-CD20 treatment. Groups of 6- to 8-week-old VH125.hCD20/NOD mice were injected with 2H7 anti-CD20 or IgG isotype control. Groups of mice ( n = 2 or 3 per group) were pooled and insulin + B cells from pancreatic islets were analysed for four different populations based on CD138 expression: CD138 − (blue); CD138 int IgM + (orange); CD138 int IgM lo (grey) and CD138 hi IgM lo (red). ( a , b ) Representative flow plots showing gating on live CD3 − CD11b − CD11c − ( a ) and graph showing the overall percentages of the four different populations ( b ). ( c , d ) Representative flow plots showing insulin − CD19 + , insulin + CD19 + and insulin + CD19 − cells ( c ) and graph showing the overall percentages of these cells ( d ); 2H7 (black circles), IgG (grey circles). ( e ) Representative flow plots showing CD138 and IgM expression in insulin + CD19 + and insulin + CD19 − cells. ( f , g ) Graphs showing CD138 and IgM populations on insulin + CD19 + ( f ) and insulin + CD19 − cells ( g ) ( n = 5 groups for control IgG treatment; n = 4 groups for 2H7 treatment). Horizontal lines represent the median values. Data represent two independent experiments. * p < 0.05 (one-way ANOVA)

Article Snippet: Female VH125.hCD20/NOD mice, 6–8 weeks of age were chosen at random to receive either anti-hCD20 antibody (clone 2H7; Bio-XCell, West Lebanon, NH, USA) or control IgG2b antibody (clone MPC-11; Bio-XCell [ , , ]), as described previously [ ].

Techniques: Injection, Control, Expressing

Journal: BMC Cancer

Article Title: Humanized anti-DEspR IgG4 S228P antibody increases overall survival in a pancreatic cancer stem cell-xenograft peritoneal carcinomatosis rat nu/nu model

doi: 10.1186/s12885-021-08107-w

Figure Lengend Snippet: DEspR modulates survival and functionality of Panc1-CSCs, cscTCs, and TCs. a DEspR-protein diagram showing epitope-1 and epitope-2 with corresponding anti-DEspR mAbs 7c5, 5g12, 6g8, hu-6g8; consensus sequences for glycosylation (green), internalization recognition sequence (navy), O-glycosylation (purple), and phosphorylation (yellow). b Representative images showing internalization of fluorescent AF568-labeled 7c5 and AF568-IgG4 isotype-control in Panc1-TCs after 1- and 2-h, and associated apoptotic (Apop.) and necroptotic (Necrop.) cell morphology changes. Chi-square test for independence, two-tailed t-test p<0.0001; 7c5-treated n=280 cells, isotype-treated n=242 cells. c Flow-cytometry showing [Top]: DEspR cell-surface expression on Panc1-TCs (65–75%) and Panc1-CSCs (60–80%) [dashed-line isotype vs. red AF-568-7c5]; [Middle]: ALDEFLUOR activity/ expression in Panc1-CSCs (68–72%) [dashed-line DEAB control vs green ALDEFLUOR] and ALDEFLUOR expression of DEspR+ Panc1-CSCs (58.2–60.4%); [Bottom] : CD133 expression on Panc1 CSCs (28–34%) [dashed-line isotype vs. green AF-488-CD133] and DEspR+/CD133+ CSCs (24.1%). d Flow-cytometry showing DEspR cell-surface expression on MiaPaca2-TCs (58–65%) and Capan-1-TCs (31–40%) [dashed-line isotype vs. green AF-488-7c5]. e Panc1-CSC tumorspheres (Bar: 100 μm) with staining of DEspR+ Panc1-CSCs from dispersed tumorspheroids. After 30-min 7c5-AF568 binding, then MoFlo-sorting and plating in low-adherence cultures, phase contrast image shows viable DEspR[−] CSCs (7c5-Tx DEspR[−]) but no spheroid-formation; in contrast to minimal to non-thriving 7c5-sorted/inhibited DEspR+ CSCs (7c5-Tx DEspR+) . f Panc1-cscTCs co-express αSMA (red) and Col1A1(green), merged (yellow) (top bar: 20 μm, bottom bar: 50 μm). g Fluorescent microscopy comparing αSMA (red) expression in control (top) vs. 6g8-treated (bottom) cscTCs. Anti-DEspR- reduced αSMA-expression in Panc1-cscTCs (14.4 ± 4.3%); non-treated controls (75.2 ± 13.7%); 6g8: n = 7 high-power-fields (HPFs); control: n = 8 HPFs, ≥40 cells/HPF, p < 0.0001 two-tailed t-test. h Anti-DEspR mAbs reduced Panc1-CSC Col1A1- secretion [6g8-treated: 97 ± 16 pg/ml, 7c5-treated: 54.5 ± 2.5 pg/ml, control: 487 ± 52 pg/ml]; n = 4 replicate wells/group, *** p < 0.001, ANOVA multiple-comparisons test. i Representative double-immunofluorescence of Panc1-CDX PPC tumor: human-specific DEspR+(red), human/rat Col1A1 + (green) cscTCs within TC-islands, DEspR+Col1A1+ co-expressing cscTCs (yellow), and DEspR[−]/Col1A1+ cscTCs and stromal cells (green) in merged panel. j Representative Masson-Trichrome (MT)-stained [LEFT, MIDDLE] and H&E-stained [RIGHT] sections of PPC tumors, showing collagen-deposition (MT:blue, H&E:bright pink) surrounding cscTCs. Bar: 50 μm

Article Snippet: The following antibodies were used: 10 μg/ml 7c5-AF568, 7c5-isotype control murine IgG2b-AF568 (R&D Systems, MAB0042), and/or anti-CD133-AF488 (Creative Biomart, NAB-2017-VHH).

Techniques: Glycoproteomics, Sequencing, Phospho-proteomics, Labeling, Control, Two Tailed Test, Flow Cytometry, Expressing, Activity Assay, Staining, Binding Assay, Microscopy, Immunofluorescence

Journal: BMC Cancer

Article Title: Humanized anti-DEspR IgG4 S228P antibody increases overall survival in a pancreatic cancer stem cell-xenograft peritoneal carcinomatosis rat nu/nu model

doi: 10.1186/s12885-021-08107-w

Figure Lengend Snippet: Humanized anti-DEspR mAb exhibits improved potency, retains DEspR/mAb internalization and nuclear translocation. a 3D-model of humanized anti-DEspR [Left] hu-6g8, [Right] mu-6g8: complementary determining regions (navy), heavy-chain (aqua), light-chain (pink). b - d Comparison of hu-6g8 and mu-6g8 b binding-affinity to intact DEspR on Panc1-TCs, c inhibition of Panc1-CSCs, and d HUVECs angiogenesis (EC 50 , IC 50 values: Table ). e Representative images of hu-6g8 and mu-6g8 angiogenesis inhibition, showing concentration-dependent decreased HUVEC tube-formation. f Confocal immunofluorescence of hu-6g8/DEspR internalization and nuclear-translocation (t = 15-min to 2-h) in Panc1 and MiaPaCa2 TCs. Bar = 15 μm. g Live-cell imaging of apoptotic changes in hu-6g8-treated Panc1-TCs. Bar = 20 μm. h Higher-magnification live-cell images of hu-6g8-treated Panc1 TCs showing normal, necroptotic, and apoptotic cell morphology. Bar = 10 μm. i , j Quantitative analysis of apoptotic (apop) and necroptotic (necrop) morphological changes in hu-6g8-treated. i Panc1-TCs ( n = 208, p < 0.0001) and j MiaPaCa2 ( n = 284, p < 0.0001) vs. isotype-control at 2 h; chi-squared test for independence, paired t-test. k , l Internalization of hu-6g8/DEspR complexes detected by AF568-labeled anti-human-IgG mAb (red), and colocalization with cytoplasmic-nuclear shuttling proteins: galectin-1 (gal1[aqua]) or galectin-3 (gal3[green]) in k Panc1 and l MiaPaCa2 TCs, after 15-min, and 4-h of hu-6g8-treatment. Bar = 20 μm. m , n Colocalization of DEspR/gal1(aqua) or DEspR/gal3(green) (Mander’s coefficient κ > 0.5) in Panc1 and MiaPaCa2 TCs in the cytoplasm ( m ) or nucleus ( n ), at 15-min and 4-h (Table ). (* p < 0.05, n = 261 and 251 TCs, Panc1 and MiaPaCa2, respectively, paired two-tail t-test)

Article Snippet: The following antibodies were used: 10 μg/ml 7c5-AF568, 7c5-isotype control murine IgG2b-AF568 (R&D Systems, MAB0042), and/or anti-CD133-AF488 (Creative Biomart, NAB-2017-VHH).

Techniques: Translocation Assay, Comparison, Binding Assay, Inhibition, Concentration Assay, Immunofluorescence, Live Cell Imaging, Control, Labeling

Journal: BMC Cancer

Article Title: Humanized anti-DEspR IgG4 S228P antibody increases overall survival in a pancreatic cancer stem cell-xenograft peritoneal carcinomatosis rat nu/nu model

doi: 10.1186/s12885-021-08107-w

Figure Lengend Snippet: Epitope-distinct murine and humanized anti-DEspR mAbs inhibit PPC dissemination-progression. a Diagram of anti-DEspR ex vivo pre-treatment design evaluating CSC tumorigenicity in Panc1-CSC subcutaneous and PPC xenograft nude-rat models. b Ex vivo pre-treatment with 5g12 (red) 200 μg/mL × 1-h ( n = 6) decreased Panc1-CSC tumorigenicity vs saline controls ( n = 8) ( p < 0.001, repeated t-test. c Ex vivo pre-treatment of Panc1-CSCs with anti-DEspR 5g12 (red, n = 8) and 6g8 (blue/ n = 8) (200 μg/ml × 1-h) increased survival vs saline control (black/ n = 15) ( p = 0.0088, log rank test). d Diagram of in vivo anti-DEspR treatment in PPC nude-rat models. e Survival analysis: PPC-females comparing saline-control ( n = 15) vs multi-dose (1 mg/kg ip 2x/wk. × 4 wks) murine anti-DEspR epitope-2 6g8 ( n = 8, p = 0.0002), epitope-1 7c5 ( n = 7, p = 0.002), and gemcitabine 26 mg/kg ( n = 7, ns). f Survival analysis: single-dose-treated PPC-females 3 wks after CSC-ip injection: saline controls ( n = 12), hu-6g8 at 3 mg/kg ( n = 8, p = 0.001) or 15 mg/kg ( n = 7, p = 0.0007), or gemcitabine 100 mg/kg ( n = 7, p = 0.0018). g Survival analysis in single-dosed PPC-males: hu-6g8 15 mg/kg/dose ( n = 6) vs saline ( n = 6, p = 0.02). Kaplan-Meier survival analyses with log rank and Holm-Sidak multiple pairwise comparisons testing (Table ). h , i Single-dose hu-6g8 pharmacokinetic analysis in PPC-females at t-21 days to match tumor burden and treatment onset to survival studies. Two-compartment analyses at intravenous-dosing: 3 mg/kg ( h ) and 15 mg/kg ( i ). T 1/2 , avg = 46.1 h, maximum retention time (MRT avg ) = 65.2 h

Article Snippet: The following antibodies were used: 10 μg/ml 7c5-AF568, 7c5-isotype control murine IgG2b-AF568 (R&D Systems, MAB0042), and/or anti-CD133-AF488 (Creative Biomart, NAB-2017-VHH).

Techniques: Ex Vivo, Saline, Control, In Vivo, Injection

Journal: BMC Cancer

Article Title: Humanized anti-DEspR IgG4 S228P antibody increases overall survival in a pancreatic cancer stem cell-xenograft peritoneal carcinomatosis rat nu/nu model

doi: 10.1186/s12885-021-08107-w

Figure Lengend Snippet: Analysis of anti-DEspR treatment effects on tumor burden, target engagement and bioeffects. a Representative post-mortem images of age-matched (57-days post-CSC injection) PPC saline-treated [Left] and hu-6g8 15 mg/kg-treated [Right] rats; hu- significantly decreased tumor burden. Comorbidities: dilated, necrotic small intestine in saline rats only (yellow➔), high omental tumor burden in saline (red➔) vs. minimal in hu-6g8 ( − − - ), cecum (*): dilated in saline but not in hu-6g8. b Target-engagement (red/magenta immunofluorescence+) 24-h after infusion on PPC tumors: [ Left ] IgG4-isotype: minimal, [ Right ] hu-6g8: high levels; DAPI+ nuclei (blue), colocalized hu-6g8/nucleus (magenta), RBC-autofluorescence (peach). c Target-bioeffects: activated Caspase-3 apoptosis DAB-staining (brown) in PPC tumors: [ Left ] omental tumors; [ Right ] tumor cells in liver [ Top ] saline control; [ Bottom ] hu-6g8 3 mg/kg iv. TC, PPC-tumor cells; HC, normal hepatocytes. Bar: 60-μm. d Representative immunohistochemistry images showing no activated Caspase-3 staining for apoptosis in normal tissues. Tumor vasculature (red➔). Bar = 100 μm. e - g Effect of hu-6g8 on hematologic cells: e neutrophils, f platelets, and g red blood cells (RBCs); no significant differences observed among saline, single-dose gemcitabine, and single-dose 3- and 15-mg/kg hu-6g8. h Significant difference (**) detected in neutrophil-lymphocyte (NL)-ratio with 15 mg/kg hu-6g8 treatment ( n = 4) vs saline ( n = 11), p = 0.009, two-way ANOVA, Bonferroni-correction

Article Snippet: The following antibodies were used: 10 μg/ml 7c5-AF568, 7c5-isotype control murine IgG2b-AF568 (R&D Systems, MAB0042), and/or anti-CD133-AF488 (Creative Biomart, NAB-2017-VHH).

Techniques: Drug discovery, Injection, Saline, Immunofluorescence, Staining, Control, Immunohistochemistry

Journal: BMC Cancer

Article Title: Humanized anti-DEspR IgG4 S228P antibody increases overall survival in a pancreatic cancer stem cell-xenograft peritoneal carcinomatosis rat nu/nu model

doi: 10.1186/s12885-021-08107-w

Figure Lengend Snippet: DEspR is expressed in human PDAC primary and metastatic tumors. a Representative immunofluorescence images of cryo-sections of normal human pancreas and human PDAC Stage-II-B tumors probed with AF568-labeled 7c5[epitope-1] murine-mAb (red) and DAPI+ nuclei (blue). Bar = 20 μm. b Representative immunofluorescence images of paraffin-embedded, antigen-retrieved sections of [Left] human normal pancreas and [Right] human PDAC primary tumors at different stages (I-IV), and metastatic (met.) tumors probed with AF568-labeled hu-6g8[epitope2] (red); DAPI nuclear-DNA stain (blue), with DIC overlay. Bar = 20 μm. c Quantitation of human tumors with > 50% vs < 50% tumor proportion score (TPS) for DEspR+ expression detected by AF568-hu-6g8, n = 133 PDAC-patient tumor-cores on tumor array. d Quantitation of DEspR expression of invading PDAC TCs in primary tumors with invasive tumor cells in stroma; n = 79 PDAC-patient tumor-cores

Article Snippet: The following antibodies were used: 10 μg/ml 7c5-AF568, 7c5-isotype control murine IgG2b-AF568 (R&D Systems, MAB0042), and/or anti-CD133-AF488 (Creative Biomart, NAB-2017-VHH).

Techniques: Immunofluorescence, Labeling, Staining, Quantitation Assay, Expressing

Journal: Glycobiology

Article Title: Base-modified UDP-sugars reduce cell surface levels of P-selectin glycoprotein 1 (PSGL-1) on IL-1β-stimulated human monocytes

doi: 10.1093/glycob/cww053

Figure Lengend Snippet: Characterization of IL-1β-induced PSGL-1 cell surface levels on human monocytes. ( A ) Human mononuclear cells were isolated from healthy donors ( n = 2) and incubated with anti-CD14-FITC to identify the monocyte population by flow cytometry (region R1). ( B ) CD14+ monocytes incubated with isotype control IgG-PE to reveal background fluorescence. ( C ) Scatter graph revealing the percentage of resting CD14+ monocytes displaying PSGL-1 (region R2). ( D ) Representative histogram of CD14+ monocytes and mean fluorescence intensity of PSGL-1 levels (anti-PSGL-1-PE). hPBMCs were either treated with media alone (white bar), 10 ng/mL recombinant human IL-1β (gray bar), or 100 ng/mL IL-1β (black bar) for 0–48 hr. Cells were harvested and co-stained with PE CD162, FITC CD14 and isotype matched controls. ( E ) Percentage of CD14+ monocytes displaying PSGL-1. ( F ) Mean fluorescent intensity (MFI) of cell surface PSGL-1 levels on monocytes. Data represent mean ± SEM. This figure is available in black and white in print and in color at Glycobiology online.

Article Snippet: Alexa Fluor 488 conjugated mouse anti-human CCR2 antibody (clone # 48607, FAB151G) and Alexa Fluor 488 conjugated mouse IgG2B isotype control antibody (clone 133303) were purchased from R&D systems (Abingdon, UK).

Techniques: Isolation, Incubation, Flow Cytometry, Control, Fluorescence, Recombinant, Staining

Journal: Poultry Science

Article Title: Characterization of chicken p53 transcriptional function via parallel genome-wide chromatin occupancy and gene expression analysis

doi: 10.1016/j.psj.2022.102164

Figure Lengend Snippet: Experimental model and design. LMH cells were transiently transfected with Flag-chp53 or empty vector p3xFLAG-CMV-7.1(vector). (A and B) Indirect immunofluorescence microscopy and flow cytometry analyses of p53 overexpression using a mouse anti-flag antibody followed by a FITC-conjugated goat anti-mouse IgG secondary antibody(green) at 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h or 12 h, 24 h, and 36 h post-transfection. Chromosomes were stained with DAPI (blue). Scale bars, 400 μm. (C) The protein expression levels of chicken p53( chp53 ) in LMH cells transiently transfected with the Flag-chp53 or vector plasmid were analyzed by Western blot using antibodies specifically against Flag (top) and β-actin (bottom). β-actin was used as a loading control. (D) LMH cells were transiently transfected with Flag-chp53 plasmid or negative control empty vector for 24 h. Cell-cycle profiles were determined by fluorescence-activated cell sorting of propidium iodide-stained cells. (E and F) LMH cells were transfected with a vector or Flag-chp53 plasmid and harvested at 24 h. (E) Chromatin immunoprecipitation sequencing ( ChIP-seq ) assays were performed with an anti-Flag antibody. IgG2b was used as a negative control. ChIP-qPCR analysis of the relative p53 occupancies in the promoters of CDKN1A and MDM2 in LMH cells. (F) The mRNA levels of CDKN1A and MDM2 were determined by RT-qPCR in LMH cells. (G) Illustration of the experimental workflow. Abbreviation: LMH, leghorn male hepatoma.

Article Snippet: Nonspecific antibody binding was blocked with 2% bovine serum albumin for 1 h, and then the samples were incubated with a mouse anti-DYKDDDDK antibody (flag epitope tag, 1 μg/mL; A00187, GenScript Corporation, Piscataway, NJ) against overexpressed chicken p53 or a mouse IgG2b isotype control antibody (1 μg/mL; 53484, Cell Signaling Technology, Boston, MA), followed by a secondary FITC-conjugated goat anti-mouse antibody (1:64 dilution; F9006, Sigma-Aldrich, St. Louis, MO).

Techniques: Transfection, Plasmid Preparation, Immunofluorescence, Microscopy, Flow Cytometry, Over Expression, Staining, Expressing, Western Blot, Control, Negative Control, Fluorescence, FACS, ChIP-sequencing, ChIP-qPCR, Quantitative RT-PCR