apc anti human nectin 4  (Miltenyi Biotec)

Journal: bioRxiv

Article Title: Ex vivo drug testing in metastatic biopsies reveals patient-specific vulnerabilities to cancer targeting and immune activating drugs

doi: 10.64898/2026.02.06.704037

Figure Lengend Snippet: ( A ) Schematic illustration of the drug response assay. Created with BioRender.com. TME, tumor microenvironment; IF, immunofluorescence. ( B ) Cellular composition of malignant pleural effusions (MPEs) from five NSCLC patients as determined by flow cytometry. ( C ) ATP-based viability of a MPE in different culture media. Each dot represents the mean ± standard deviation (shaded envelope) of n = 5 technical replicates from one representative donor (P179). ( D ) Flow cytometric analysis of MPE viability in HPLM medium. Each dot represents an individual donor (n = 5). P-values were calculated using Wilcoxon matched-pairs signed rank test. ( E ) Log2-fold change (Log2FC) in cellular composition of MPEs after 5 days in HPLM medium compared to baseline, analyzed by flow cytometry. Each dot represents an individual donor. Samples from n = 5 donors were measured. To illustrate fold changes, cell fractions below the 2%-detection limit were excluded from analysis. P-values were determined using a one-sample Wilcoxon test against zero. ( F ). As for (E), but for marker expression of cancer cells (PD-L1, Nectin-4, TROP2) and T cells (PD-1). ( G ) Cytokine secretion and immune checkpoint expression of MPEs (n = 5) after 5 days of ex vivo culture. Color scale represents Z-scores normalized across patients for each cytokine or marker. nMFI, mean fluorescence intensity normalized to fluorescence minus one (FMO) control. ( H ) Relative cell loss of two non-adherent cell lines following the optimized IHC liquid handling protocol. Dots represent outliers among technical replicate wells across n = 3 biological replicates (384-well plates). P-values were calculated using a one-sample Wilcoxon test. ( I ) Pseudocolor plot depicting MFIs of CD45 and EpCAM signal for all segmented masks of a spike-in of MCF-7 cells in lymph node cells. Cell populations were classified based on manual gating. ( J ) Number of cancer (left) and immune cells (right) detected using our image analysis pipeline. Box plots represent data of n = 10 technical replicate wells. ( K ) Cancer cell fractions in malignant pleural effusions (MPEs) from NSCLC patients (n = 5), determined by EpCAM-based immunocytochemistry. The dashed line indicates the detection limit (10 cells per well) of the drug response assay.

Article Snippet: Cells were resuspended in 100 μL of FACS buffer (1% (w/v) BSA, 2 mM EDTA in DPBS) per 1 x 10 6 cells and stained using the following antibodies: Alexa Fluor® 488 anti-human CD3, clone HIT3a (BioLegend, #300319, RRID:AB_493690), PerCP/Cyanine5.5 anti-human CD14, clone HCD14 (BioLegend, # 325621, RRID:AB_893252), PE anti-human CD279 (PD-1), clone EH12.2H7 (BioLegend, # 329905, RRID:AB_940481), PE/Cyanine7anti-human CD11c, clone Bu15 (BioLegend, # 337215, RRID:AB_2129791), APC-Vio770 anti-human CD19, clone LT19 (1:50; Miltenyi Biotec, # 130-098-073, RRID:AB_2661296), Brilliant VioletTM 421 anti-human CD45, clone 2D1 (BioLegend, # 368522, RRID:AB_2687375), Brilliant VioletTM 605 anti-human HLA-DR, clone L243 (BioLegend, # 307639, RRID:AB_11219187), Brilliant VioletTM 785 anti-human CD56 (NCAM), clone 167 (BioLegend, # 362549, RRID:AB_2566058), Alexa Fluor® 488 anti-human CD326 (EpCAM), clone Co17-1A (BioLegend, # 369808, RRID:AB_2650905), PE anti-human TROP2, REAfinityTM (1:50; Miltenyi Biotec, # 130-115-097, RRID:AB_2726914), APC anti-human Nectin-4, REAfinityTM (1:50; Miltenyi Biotec, # 130-116-103, RRID:AB_2727350), Brilliant VioletTM 421 anti-human CD274 (B7-H1, PD-L1), clone 29E.2A3 (BioLegend, # 329714, RRID:AB_2563852), Brilliant VioletTM 785 anti-human CD45, clone HI30 (BioLegend, # 304048, RRID:AB_2563129), Zombie AquaTM (1:1,000; BioLegend, # 423102), Zombie NIRTM (1:500; BioLegend, # 423106).

Techniques: Immunofluorescence, Flow Cytometry, Standard Deviation, Marker, Expressing, Ex Vivo, Fluorescence, Control, Immunocytochemistry

Journal: Virology

Article Title: The V domain of dog PVRL4 (nectin-4) mediates canine distemper virus entry and virus cell-to-cell spread.

doi: 10.1016/j.virol.2014.02.014

Figure Lengend Snippet: Fig. 1. Amino acid sequences of all human PVRL molecules and dog PVRL4 in the V domain. (A) Sequence alignment of the V domain from human PVRL1 to PVRL4, human PVR, and dog PVRL4. Residues having similarity are shaded (dark shading represents identical amino acid residues; dark gray shading represents residues with 80–100% identity, light gray shading represents residues with 60–80% identity, no shading represents residues with less than 60% identity). The consensus FPxG amino acid sequence is boxed in red. Pairwise protein alignments were performed using Geneious sequence alignment software (Drumond et al., 2010). (B) Schematic diagrams of the wild-type and chimeric dog PVRL4 and human PVRL1 proteins (dog.PVRL4, dog.PVRL4/VhPVRL1, hPVRL1 and hPVRL1/Vdog.

Article Snippet: Briefly, cells were washed with PBS, non-enzymatically dissociated and subsequently blocked in 2.5 μg of normal human IgG (R&D Systems) for 10 min on ice followed by the addition of 10 μl of either PE-conjugated monoclonal antibody against human PVRL4 27-351 (R&D Systems FAB2659P) or PE-conjugated mouse IgG2B isotype control (R&D Systems IC0041P), and APC-conjugated mouse monoclonal antibody against human PVRL1 31-334 (R&D Systems FAB2880A) or APC-conjugated mouse IgG2A isotype control (R&D Systems IC003A) antibodies for 45 min on ice.

Techniques: Sequencing, Software

Journal: Virology

Article Title: The V domain of dog PVRL4 (nectin-4) mediates canine distemper virus entry and virus cell-to-cell spread.

doi: 10.1016/j.virol.2014.02.014

Figure Lengend Snippet: Fig. 2. Cell surface expression of chimeric PVRL molecules (dog.PVRL4, dog.PVRL4/VhPVRL1 and hPVRL1/Vdog.PVRL4). The flow cytometric histograms depict the cell surface expression profile of PVRL4 and PVRL1 in the corresponding cell line (isotype ctrl antibody, IgG – shaded; PVRL4 antibody, PVRL4 – blue; PVRL1 antibody, PVRL1 – green). Vero stable cell lines were incubated with either a phycoerythrin (PE)-conjugated mouse monoclonal antibody specific for human PVRL4, a PE-conjugated mouse IgG2a control antibody, an allophycocyanin (APC)-conjugated mouse monoclonal antibody specific for human PVRL1 or an APC-conjugated mouse IgG2B control antibody. The Y-axis represents cell counts and the X-axis represents fluorescence intensity (FL8: APC; FL2: PE).

Article Snippet: Briefly, cells were washed with PBS, non-enzymatically dissociated and subsequently blocked in 2.5 μg of normal human IgG (R&D Systems) for 10 min on ice followed by the addition of 10 μl of either PE-conjugated monoclonal antibody against human PVRL4 27-351 (R&D Systems FAB2659P) or PE-conjugated mouse IgG2B isotype control (R&D Systems IC0041P), and APC-conjugated mouse monoclonal antibody against human PVRL1 31-334 (R&D Systems FAB2880A) or APC-conjugated mouse IgG2A isotype control (R&D Systems IC003A) antibodies for 45 min on ice.

Techniques: Expressing, Stable Transfection, Incubation, Control

Journal: Virology

Article Title: The V domain of dog PVRL4 (nectin-4) mediates canine distemper virus entry and virus cell-to-cell spread.

doi: 10.1016/j.virol.2014.02.014

Figure Lengend Snippet: Fig. 3. CDV 5804PeH replication in wild-type, chimeric dog PVRL4 and human PVRL1-expressing Vero stable cell lines. Vero.dog.PVRL4, Vero.dog.PVRL4/VhPVRL1, Vero. hPVRL1/Vdog.PVRL4, and Vero cells were seeded on twelve-well plate (1 105 cells/well) 1 day before infection with CDV 5804PeH at an MOI of 0.5. (A) Fluorescence images were captured three days post infection and overlaid with phase contrast images to visualize the extent of virus replication (GFP) and syncytia formation. (B) CDV-infected cells were harvested at 3 days post-infection and titered on Vero.dog.SLAM cells. Data are the means from three independent experiments, and error bars represent the SEM. Analysis of variance (ANOVA) was performed to identify statistically significant differences in CDV 5804PeH titrations. P values below 0.05 were considered statistically significant.

Article Snippet: Briefly, cells were washed with PBS, non-enzymatically dissociated and subsequently blocked in 2.5 μg of normal human IgG (R&D Systems) for 10 min on ice followed by the addition of 10 μl of either PE-conjugated monoclonal antibody against human PVRL4 27-351 (R&D Systems FAB2659P) or PE-conjugated mouse IgG2B isotype control (R&D Systems IC0041P), and APC-conjugated mouse monoclonal antibody against human PVRL1 31-334 (R&D Systems FAB2880A) or APC-conjugated mouse IgG2A isotype control (R&D Systems IC003A) antibodies for 45 min on ice.

Techniques: Expressing, Stable Transfection, Infection, Fluorescence, Virus

Journal: Virology

Article Title: The V domain of dog PVRL4 (nectin-4) mediates canine distemper virus entry and virus cell-to-cell spread.

doi: 10.1016/j.virol.2014.02.014

Figure Lengend Snippet: Fig. 4. Characterization of CDV glycoproteins-induced cell–cell fusion in wild-type, chimeric dog PVRL4 and human PVRL1-expressing Vero stable cell lines. Vero.dog.PVRL4, Vero.dog.PVRL4/VhPVRL1, Vero.hPVRL1/Vdog.PVRL4, Vero.dog.SLAM, and Vero cells were cotransfected with plasmid encoding CDV F together with CDV H or CDV HP493S/Y539A. At 48 h post transfection, cells were stained with Giemsa as described in Materials and methods. Light microscopy images were captured to visualize the extent of syncytia formation. Arrow heads indicate the location of representative syncytia in transfected cells.

Article Snippet: Briefly, cells were washed with PBS, non-enzymatically dissociated and subsequently blocked in 2.5 μg of normal human IgG (R&D Systems) for 10 min on ice followed by the addition of 10 μl of either PE-conjugated monoclonal antibody against human PVRL4 27-351 (R&D Systems FAB2659P) or PE-conjugated mouse IgG2B isotype control (R&D Systems IC0041P), and APC-conjugated mouse monoclonal antibody against human PVRL1 31-334 (R&D Systems FAB2880A) or APC-conjugated mouse IgG2A isotype control (R&D Systems IC003A) antibodies for 45 min on ice.

Techniques: Expressing, Stable Transfection, Plasmid Preparation, Transfection, Staining, Light Microscopy