Human/Mouse Arginase 1/ARG1 Fluorescein‑conjugated Antibody

Detection of Human Arginase 1/ARG1/liver Arginase by Flow Cytometry

Ultracentrifugated (UC) GSC-derived exosomes promote an immunosuppressive phenotype in monocytes similarly to ExoQuick (EQ) purified GSC- or GBM-derived exosomes.(A) CFSE-labelled PBMCs isolated from healthy donors (left) or CD14 negatively-sorted PBMCs (CD14-) (right) were pre-treated for 24 hours without (white column, CTRL) or with EQ or UC isolated GSC-derived exosomes (black column, EQ GSC-EXO; grey column, UC GSC-EXO, respectively) and stimulated for 4 days with anti-CD3 and anti-CD28. Histograms show a significant difference in the percentage of proliferating CD3+ (n = 4); bars, SD;*; from the control; P<0.05. (B-D) Induction of a Mo-MDSC phenotype on monocytes. Unstimulated PBMCs were incubated in the absence (CTRL) or presence of GSC-derived exosomes purified by EQ (black column, EQ EXO-GSC) or UC(grey column, UC EXO-GSC). Cells were surface stained with (B) anti-CD14, anti CD33, anti CD11b and (C) HLA-DR and then stained to detect intracellular level of IL-10 and arginase-1 by flow cytometry. (B) Gating strategy: physical parameters, i.e. forward scatter (FSC) and side scatter (SSC), were used to select monocytes (gate R1, left panel). Monocytes were recognized by evaluating the expression of CD11b/CD33 (gate R2, middle panel) and CD14/HLA-DR (gate R3, right panel). (C) Representative FACS histograms of the intracellular staining of IL-10 and arginase-1 and of HLA-DR staining of CD14+/CD11b/CD33+ cells are shown. (D) The percentage of cells expressing IL-10 and arginase-1 and the MFI ratio of HLA-DR expression are shown (n = 3); bars, SD;*, significantly different from the control; *, P<0.05; **,P<0.01. (E) PBMCs were stimulated with anti-CD3 and anti-CD28 in the absence (white column, CTRL) or presence (black column, GBM-EXO) of exosomes isolated from plasma of glioblastoma patients (GBM) by either EQ or UC and used at 1:10 dilution. Healthy donor plasma-derived exosomes were used as a control (striped column, EXO-HEALTHY). Proliferation of CD3+ was measured by CFSE assay at day 4. Proliferation in the presence of exosomes was normalized to CD3+ cell proliferation in the absence of exosomes (CTRL set to 100%). (F) Proliferation of both unfractioned PBMCs and PBMCs depleted of the CD14+ population (CD14-) was measured, after CFSE labelling assay, by flow cytometry. Cells were pre-incubated without (white column, CTRL) or with EQGBM-derived exosomes (black column, EQ GBM-EXO) or UC GBM-derived exosomes (grey column, UC GBM-EXO). Columns, mean (n = 4); bars, SD; *, significantly different from the control; P<0.05. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0169932), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Arginase 1/ARG1/liver Arginase by Flow Cytometry

Arginase 1 expression in different subsets of circulating MDSC in patients with PC. Flow cytometric evaluation of ARG1 expression in CD33, CD11b, CD15 and CD14 in whole blood is shown in the representative dot plots. Gates were set based on negative controls. Numbers represent the percentages from the original populations gated. P (number) above each FACS plot indicates the population gated which was analysed. The gate was first set on HLA-DR negative against side scatter as shown in the top dot plot. Next, the CD11b+ & CD33+ (right) and CD14+ & CD15+ (left) subsets were identified. The expression of ARG1 in each subset was then determined as shown in the bottom dot plots. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/24741628), licensed under a CC-BY license. Not internally tested by R&D Systems.