cxcr2 tango plasmid  (Addgene inc)

Journal: Nature Communications

Article Title: Selection of a picomolar antibody that targets CXCR2-mediated neutrophil activation and alleviates EAE symptoms

doi: 10.1038/s41467-021-22810-z

Figure Lengend Snippet: a Representative images of co-localization of abN48-IgG1 or abN48-2-IgG1 to h CXCR2, h CXCR1, and m CXCR2 overexpressed on the membrane of U2OS cells. All test receptor proteins were fused with an mCherry fluorescent tag, and the immunocytofluorescent images were captured by confocal microscopy (3 images of every 3 samples were analyzed for each group and all gave consistent results). Scale bars (white) represent 50 μm. b Surface interactions of abN48-IgG1 and abN48-2-IgG1 with h CXCR2, h CXCR1, m CXCR2, r CXCR2, rb CXCR2, and mc CXCR2 overexpressed on U2OS cells. Flow-cytometry was used to measure the interactions between antibodies and the CXCR2 receptor proteins. Isotype-antibody (irrelevant human IgG1 antibody) was used as a negative control. For each sample, 3 repeats were carried out and all gave consistent results.

Article Snippet: Tango TM CXCR2-bla U2OS cell line (#K1807; Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Membrane, Confocal Microscopy, Flow Cytometry, Negative Control

Journal: Nature Communications

Article Title: Selection of a picomolar antibody that targets CXCR2-mediated neutrophil activation and alleviates EAE symptoms

doi: 10.1038/s41467-021-22810-z

Figure Lengend Snippet: a Activation of β-arrestin signaling by IL-8, GRO-α, and the antibody ligands using a Tango reporter gene assay. b Inhibition of chemokine-induced β-arrestin signaling by abN48-IgG1 and abN48-2-IgG1. The induction concentration of IL-8 (left) and GRO-α (right) were at their corresponding EC 80 values. c Activation of Ca 2+ influx by IL-8, GRO-α, and the abN48 antibody ligands using FLIPR measurement. d Inhibition of chemokine-induced Ca 2+ influx by abN48-IgG1 and abN48-2-IgG1. The induction concentration of IL-8 (left) and GRO-α (right) were at their corresponding EC 90 values. Data are represented as mean ± standard deviation. The calculated EC 50 and IC 50 values are listed next to the fitted curves. (For Tango and Ca 2+ assay ( a – d ), n = 3 independent reporter cell samples measured in a single experiment, data presented as mean (center) ± s.d. (error bars). Each assay was repeated 3 times and all gave consistent results). e Antibody-dependent CXCR2 internalization. U2OS( + ) and U2OS(−) represent U2OS cells with and without surface h CXCR2 expression, respectively; CT represents the cytotoxin AL1-PE38KDEL; abN48 represents the antibody without cytotoxin; IT-iso, IT-1, and IT-2 represent the immunotoxins (ITs) assembled with an irrelevant human IgG1 antibody, abN48-IgG1 and abN48-2-IgG1 to the cytotoxin AL1-PE38KDEL, respectively. ( n = 3 independent cell samples tested in a single experiment, data presented as mean (center) ± s.d. (error bars). This assay was repeated twice and gave consistent results.).

Article Snippet: Tango TM CXCR2-bla U2OS cell line (#K1807; Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Activation Assay, Reporter Gene Assay, Inhibition, Concentration Assay, Standard Deviation, Expressing

Journal: Frontiers in Immunology

Article Title: Defective Localization With Impaired Tumor Cytotoxicity Contributes to the Immune Escape of NK Cells in Pancreatic Cancer Patients

doi: 10.3389/fimmu.2019.00496

Figure Lengend Snippet: Chemokines and their receptor expression in PDAC patients. (A) qRT-PCR analysis of multiple chemokine expression in tumor tissues and non-tumor tissues harvested from patients undergoing resection surgery. (B) Flow cytometry analysis of the expression of chemokine receptors in circulating NK cells from patients vs. that from healthy donors. (C) CXCR2 expression on NK cells in healthy donor was gated on CD56hi and CD56lo populations (left), and their percentages in HD and PDAC patients are summarized as a table (right), with the average level of CXCR2 expression on CD56hi and CD56lo NK cells shown as bar graphs (D) Flow cytometry analysis of CXCR2 expression on NK cells from PBMC and TIL of patients with PDAC. (E) Schematic diagrams of lentivirus construction expressing CXCR2 gene; CXCR2 gene was cloned upstream of RSV-copGFP cassette in the transfer vector pCDH-521A. The level of CXCR2 expression on transduced NK cells was shown by FACs. (F) Migration assay was performed using vector-or CXCR2-transduced patients' NK cells targeting MIA PaCa-2 (left) or Patients' tumor cells (right). Bar graph shows the percentage of NK cells migrated toward tumor targets. Data shown here are representative of three independent experiments. Statistical significance was determined by one-way ANOVA ( * p ≤ 0.05; *** p < 0.001).

Article Snippet: Full-length human CXCR2 was obtained from Addgene (cat #66260, MA) and amplified by polymerase chain reaction. pCDH-521A lentiviral plasmids carrying CXCR2 were co-transfected with packaging plasmids into 293 FT cells using CaCl 2 , and supernatant was collected after 72 h of culture.

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, Clone Assay, Plasmid Preparation, Migration