anti human pd1  (Cell Signaling Technology Inc)

Journal: Frontiers in Immunology

Article Title: PD-L1 targeted peptide demonstrates potent antitumor and immunomodulatory activity in cancer immunotherapy

doi: 10.3389/fimmu.2024.1367040

Figure Lengend Snippet: The physicochemical properties of PPL-C peptides. PPL-C binding specificity to hPD-L1, mPD-L1, and BSA was analyzed by fluorescence-based ELISA ( A , n = 3). Ligand inhibition assay analysis of the changes in fluorescence signals of PPL-C-FITC (green and blue lines) and PD-1-PE (red lines) with the increased concentration of peptide was analyzed by fluorescence-based ELISA ( B , n = 3). The binding ability of PPL-C (blue lines) or PD-L1 mAb (red lines) to CHO (gray) and CHO-PD-L1 cells was determined by flow cytometry ( C , n = 3). Statistical analysis of the results of three repeated flow experiments, where Pos% is the fluorescence positive rate, and Mean and Median are the mean fluorescence intensity values and median fluorescence intensity values ( D , n = 3). PD-L1 expression on CT26 was detected by flow cytometry (E) . Different concentrations of PPL-C/FITC peptides and a single concentration of biotinylated PD-1 were incubated simultaneously to detect PD-1 binding capacity on LLC, CT26 and MGC-803 cell lines (F) . Data, mean ± SEM; *,P < 0.05; **,P < 0.01.

Article Snippet: Human PD-L1 natural ORF mammalian expression plasmid (Cat: HG10084-UT), rabbit anti-human PD-1 antibody (Clone D67, Cat: 10377-RD67), recombinant human PD-L1 protein (Cat: 10084-H08H), human PD-L1/B7-H1/CD274 protein (Cat: 10084-HNAH), biotinylated mouse PD-1/PDCD1 protein (His tag) and recombinant human PD-1 protein (Cat: 10377-H08H) were purchased from Sino Biological Inc (Beijing).

Techniques: Binding Assay, Fluorescence, Enzyme-linked Immunosorbent Assay, Inhibition, Concentration Assay, Flow Cytometry, Expressing, Incubation

Journal: Nanomaterials

Article Title: Engineering Cell-Derived Nanovesicles for Targeted Immunomodulation

doi: 10.3390/nano13202751

Figure Lengend Snippet: Generation and characterization of PD1-expressing CDNVs. ( A ) A-MSCs were transduced with membrane GFP (mGFP) or PD1-mGFP lentivirus. ( B ) Total PD1 expression in mGFP or PD1-mGFP transduced A-MSCs was confirmed by Western blot, and ( C ) PD1 on their surface was evaluated using flow cytometry. Control CDNVs and PD1+ CDNVs isolated from mGFP and PD1-mGFP transduced A-MSCs, respectively, were characterized for particle size by ( D ) NTA and ( E , F ) PD1 expression on their surface by ImageStream flow cytometry. ( G ) HuCCT1 cells were incubated with 1 × 10 9 control of PD1+ CDNVs for 24 h, and viable cells were assessed using an MTS assay. The values are expressed as the mean ± standard deviation (n = 3 replicates). Statistically significant data were represented as follows: ***, p < 0.001.

Article Snippet: DiO labeled CDNVs were then incubated with rabbit anti-human PD1 antibody (Cell Signaling Technology, Danvers, MA, USA) for 1 h at RT, followed by incubation with anti-rabbit AlexaFlour647 secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 1 h at RT.

Techniques: Expressing, Transduction, Membrane, Western Blot, Flow Cytometry, Control, Isolation, Incubation, MTS Assay, Standard Deviation

Journal: Nanomaterials

Article Title: Engineering Cell-Derived Nanovesicles for Targeted Immunomodulation

doi: 10.3390/nano13202751

Figure Lengend Snippet: PD1-expressing CDNVs bind to PD-L1 and exhibit enhanced uptake efficiency. HuCCT1 cells were incubated with 1 × 10 9 and 1 × 10 10 DiO-labeled control or PD1+ CDNVs for binding and uptake studies, respectively. ( A ) The HuCCT1 cells were stained with PD-L1 antibody (AF647) and DAPI (nucleus), and images were captured using the confocal microscope; the scale bar represents 25 µm. For uptake studies, imaging-based flow cytometry was performed using ImageStream. ( B ) The DiO-stained CDNVs were detected in channel Ch02, and the viability dye was visualized in channel Ch03. IDEAS software v6.2 was used to extract .fcs files, and the mean fluorescence intensity (MFI) was calculated in FlowJo v10.8.1. The values are expressed as the mean ± standard deviation (n = 3 replicates). Statistically significant data were represented as follows: ***: p < 0.001.

Article Snippet: DiO labeled CDNVs were then incubated with rabbit anti-human PD1 antibody (Cell Signaling Technology, Danvers, MA, USA) for 1 h at RT, followed by incubation with anti-rabbit AlexaFlour647 secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 1 h at RT.

Techniques: Expressing, Incubation, Labeling, Control, Binding Assay, Staining, Microscopy, Imaging, Flow Cytometry, Software, Fluorescence, Standard Deviation

Journal: Nanomaterials

Article Title: Engineering Cell-Derived Nanovesicles for Targeted Immunomodulation

doi: 10.3390/nano13202751

Figure Lengend Snippet: Biodistribution of PD1-expressing CDNVs. ( A , B ) Tumor-bearing mice were intravenously injected with DiR labeled 1 × 10 10 control CDNVs or PD1+ CDNVs. After 6 h, organs were collected, and the fluorescence was measured ex situ using an IVIS imaging system.

Article Snippet: DiO labeled CDNVs were then incubated with rabbit anti-human PD1 antibody (Cell Signaling Technology, Danvers, MA, USA) for 1 h at RT, followed by incubation with anti-rabbit AlexaFlour647 secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 1 h at RT.

Techniques: Expressing, Injection, Labeling, Control, Fluorescence, Ex Situ, Imaging

Journal: Nanomaterials

Article Title: Engineering Cell-Derived Nanovesicles for Targeted Immunomodulation

doi: 10.3390/nano13202751

Figure Lengend Snippet: PD1-expressing CDNVs potentiate T cell-mediated cytotoxicity. HuCCT1 cells were pretreated with 1 × 10 10 control or PD1+ CDNVs for 3 h and 6 h for degranulation and cytotoxicity experiments, respectively. CDNV pretreated HuCCT1 cells were incubated with T cells in an effector: target (E:T) ratio of 5:1 or 10:1 to assess ( A , B ) the degranulation activity of T cells and ( C ) their cytotoxicity on cancer cells, relative to untreated control. The values are expressed as the mean ± standard deviation (n = 3 replicates). Statistically significant data were represented as follows: *, p < 0.05; **, p < 0.01.

Article Snippet: DiO labeled CDNVs were then incubated with rabbit anti-human PD1 antibody (Cell Signaling Technology, Danvers, MA, USA) for 1 h at RT, followed by incubation with anti-rabbit AlexaFlour647 secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 1 h at RT.

Techniques: Expressing, Control, Incubation, Activity Assay, Standard Deviation

Journal: Nanomaterials

Article Title: Engineering Cell-Derived Nanovesicles for Targeted Immunomodulation

doi: 10.3390/nano13202751

Figure Lengend Snippet: PD1-expressing CDNVs potentiate NK cell-mediated cytotoxicity. HuCCT1 cells were pretreated with 1 × 10 10 control or PD1+ CDNVs for 3 h and 6 h for degranulation and cytotoxicity experiments, respectively. CDNV-pretreated HuCCT1 cells were incubated with NK cells in an effector to target (E:T) ratio of 5:1 or 10:1 to assess ( A , B ) the degranulation activity of NK cells and ( C ) their cytotoxicity on cancer cells, normalized to the untreated control. The values are expressed as the mean ± standard deviation (n = 3 replicates). Statistically significant data were represented as follows: **, p < 0.01; ***, p < 0.001.

Article Snippet: DiO labeled CDNVs were then incubated with rabbit anti-human PD1 antibody (Cell Signaling Technology, Danvers, MA, USA) for 1 h at RT, followed by incubation with anti-rabbit AlexaFlour647 secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 1 h at RT.

Techniques: Expressing, Control, Incubation, Activity Assay, Standard Deviation