Journal: bioRxiv

Article Title: ARG1 could be expressed by human, but not represent for repair

doi: 10.1101/2020.07.02.183624

Figure Lengend Snippet: (A) The schematic diagram shows the time point after the pre-operation of GBR when tissues were harvested and the assays performed. (B and C) Analysis of RNA-seq data: both fpkm (B) and gene count (C) confirmed that ARG1 was not detected in the normal and inflammatory states, and its expression was low at the healing site, even when compared with other genes related to macrophages. (D) qPCR assay of ARG1 showed no significant differences between tissues from inflammatory and healing sites. Tissues were collected and digested to obtain single-cell suspensions for flow cytometry analysis. CD68and CD11b were used as markers for myeloid macrophages. iNOS and ARG1 were used as markers for M1 and M2 macrophages. (E) ARG1 expression was not detected in either group, and CD11b + CD68 + ARG1 + iNOS − macrophages were not observed using gated and t-distributed stochastic neighbor embedding (t-SNE) dimension reduction to visualize high-dimensional data. (F) Immunofluorescent staining did not reveal ARG1 in any group. Hoechst staining appears as blue in color while ARG1 appears as red. (G) Detection of surface markers by flow cytometry analysis. High expression of CD68 (CD68-FITC, Clone Y1/82A) and low expression of Arg1 were evident in the three groups. The population size of CD11b + CD68 + ARG1 − iNOS + macrophages was very small. Abbreviations: NB, normal alveolar bone tissue; IP, inflammatory periodontal tissue; H, healing (tissue harvest after guided bone regeneration surgery), and N/A, undetected.

Article Snippet: Cells were isolated by trypsinization after cultured for 1 (THP-1 monocytes to macrophages) and 7 (THP-1 monocytes derived macrophages after culturing in different circumstances) days respectively, co-incubated with antibodies against iNOS (iNOS-PE, Clone 4E5, Novus Biologicals, USA), CD68 (CD68-FITC, Clone Y1/82A, Abcam, UK), CD11b (PerCP/Cy5.5®, Clone M1/70, Abcam, UK) and ARG1 (ARG1-PE/Cyanine, Clone 14D2C43, BioLegend, USA) at 1:400 dilution in the dark for 1 h at 4°C (100μl per antibody for each sample).

Techniques: RNA Sequencing, Expressing, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: ARG1 could be expressed by human, but not represent for repair

doi: 10.1101/2020.07.02.183624

Figure Lengend Snippet: (A) Schematic diagram of the cell culture process and the time points for stimulation of macrophage differentiation, seeding macrophages in the different conditions, and performing the assays. (B) Light microscopy images of macrophages cultured under different conditions on Day 7. (C) Overlay of tSNE dimensionality reduction diagrams revealed the absence of AGR1 + macrophages in the flow cytometry analysis. (D) qPCR assay of ARG1 using Gapdh as the reference gene did not detect ARG1 in the four groups. (E-G) In contrast to the large population of CD11b + CD68 + macrophages (E) and CD68 + CD11b + iNOS + ARG − (F), CD68 + CD11b + iNOS − ARG1 + macrophages were not detected (G). Abbreviations: Ctrl, RPMI supplemented with 10% FBS; LPS, RPMI supplemented with 10% FBS and 10 ng/ml LPS; IL4, RPMI supplemented with 10% FBS, 10 ng/ml IL4; DBBM, RPMI supplemented with 10% FBS and 0.03 g/well DBBM; NS, no significance; N/A, undetected. *** denotes P < 0.001.

Article Snippet: Cells were isolated by trypsinization after cultured for 1 (THP-1 monocytes to macrophages) and 7 (THP-1 monocytes derived macrophages after culturing in different circumstances) days respectively, co-incubated with antibodies against iNOS (iNOS-PE, Clone 4E5, Novus Biologicals, USA), CD68 (CD68-FITC, Clone Y1/82A, Abcam, UK), CD11b (PerCP/Cy5.5®, Clone M1/70, Abcam, UK) and ARG1 (ARG1-PE/Cyanine, Clone 14D2C43, BioLegend, USA) at 1:400 dilution in the dark for 1 h at 4°C (100μl per antibody for each sample).

Techniques: Cell Culture, Light Microscopy, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: TGFβ-derived immune modulatory vaccine: targeting the immunosuppressive and fibrotic tumor microenvironment in a murine model of pancreatic cancer

doi: 10.1136/jitc-2022-005491

Figure Lengend Snippet: Vaccination with TGFβ-derived peptides increases the tumoral-infiltration of CD8 + T cells and polarizes tumor-associated macrophages from an M2-like to an M1-like phenotype. Pan02 tumor-bearing mice (n=8 per group) were either left untreated or vaccinated with the TGFβ vaccine on days 10 and 17. Tumors were harvested on day 33, pooled in pairs among treatment groups, and analyzed by flow cytometry. Bar plots show (A) cancer cells gated as CD45 − CD31 − FAP − , (B) leukocytes gated as CD45 + CD31 − , (C) endothelial cells gated as CD45 − CD31 + , (D) CD3 + T cells gated as CD45 + CD3 + , (E) CD8 + T cells gated as CD45 + CD3 + CD8 + CD4 − , (H) CD4 + T cells gated as CD45 + CD3 + CD8 − CD4 + , (I) CD8/CD4 ratio calculated by dividing the percentage of CD8 + T cells among CD3 + cells by the percentage of CD4 + T cells among CD3 + cells, (J) Tregs gated as CD45 + CD3 + CD8 − CD4 + CD25 + FoxP3 + , (K) CD8/Treg ratio calculated by dividing the percentage of CD8 + T cells among CD3 + cells by the percentage of Tregs among CD3 + cells, (L) macrophages gated as CD11b + F4/80 + , (M) M1 macrophages gated as CD11b + F4/80 + mannose receptor (MR) − , (N) M2 macrophages gated as CD11b + F4/80 + MR + and (O) M1/M2 ratio calculated by dividing the percentage of M1 macrophages by the percentage of M2 macrophages in the tumor of untreated or mice treated with the TGFβ vaccine. All populations were gated on single live cells. Gating strategy can be found in . Data are presented as mean±SEM. Dots represent pooled tumors (n=3–4 per group). (F) and (G) Representative dot plots of CD4 + and CD8 + cells in the CD3 + population of (F) untreated or (G) TGFβ vaccine-treated mice. (P) Representative histograms of MR on macrophages in untreated mice or mice treated with the TGFβ vaccine shown in (M) and (N). ns, not significant; *p<0.05 and **p<0.01 according to unpaired two-tailed t test. TGFβ, transforming growth factor-β; Tregs, regulatory T cells.

Article Snippet: The following antibodies/dies (purchased from BioLegend unless otherwise stated) were used for flow cytometry: CD45-PE-Cy7, CD31-FITC, FAP-biotin (R&D system), streptavidin-APC, CD90-BV605, PDPN-APC, Ly6C-AF700, CD26-PerCP-Cy5.5 (eBioscience), αSMA-Cy3 (Sigma), CD11b-PE-Cy7, CD11b-Pacific Blue, F4/80-APC, F4/80-FITC, MR-PE, MR-PE-Cy7, Ly6C-PerCP-Cy5.5, Ly6G-APC-Cy7, Arg1-PE (R&D system), PDL1-APC (BD Biosciences), CD45-FITC, CD3-AF700, CD4-BV421, CD8-BV605 (BD Biosciences), CD25-PE-Cy7, FoxP3-APC and carboxyfluorescein succinimidyl ester (CFSE, Sigma Aldrich).

Techniques: Derivative Assay, Flow Cytometry, Two Tailed Test

Journal: Journal for Immunotherapy of Cancer

Article Title: TGFβ-derived immune modulatory vaccine: targeting the immunosuppressive and fibrotic tumor microenvironment in a murine model of pancreatic cancer

doi: 10.1136/jitc-2022-005491

Figure Lengend Snippet: TGFβ vaccination results in reduced intratumoral secretion of TGFβ and reduces immunosuppression of T cells and macrophages in the tumor microenvironment. Pan02 tumor-bearing mice were either left untreated or vaccinated with the TGFβ vaccine on days 10 and 17. Tumors were harvested (n=4 per group) on day 24 and pooled in pairs among treatment groups. Tumor-conditioned media (TCM) secreted by 0.1×10 6 cells from the tumor digest during a 48 hours culture was harvested. (A) Quantification of TGFβ1 protein levels in TCM from untreated and TGFβ-vaccinated mice. (B) Effect of TCM from untreated or TGFβ-vaccinated mice on the proliferation of anti-CD3/CD28-stimualted naïve splenocytes from an untreated tumor-free mouse. Naïve splenocytes were CFSE-labeled and activated with Dynabeads Mouse T-Activator CD3/CD28 (1:1 ratio) for 48 hours in the presence or absence of TCM generated from tumors from untreated or TGFβ-vaccinated mice. Proliferation of live CD3 + cells was measured by flow cytometry. Gating strategy can be found in . (B, left) Proliferation index in CD3 + cells across culture conditions. (B, right) Representative histograms of CFSE staining in live CD3 + cells (B). (C–E) Effect of TCM from untreated or TGFβ-vaccinated mice on the phenotype of bone-marrow derived macrophages (BMDM) from an untreated tumor-free mouse that were polarized with IL-4 to an M2-like phenotype. BMDM were cultured for 24 hours in the presence of TCM generated from tumors from untreated or TGFβ-vaccinated mice. The phenotype of BMDM was assessed by flow cytometry. (C, top) M1/M2 ratio in BMDM cultured with TCM generated from tumors from untreated or TGFβ-vaccinated mice. Macrophages were gated as CD11b + F4/80 + , M1 and M2 macrophages were gated as CD11b + F4/80 + mannose receptor (MR) − and CD11b + F4/80 + MR + , respectively. Gating strategy can be found in . (C, bottom) Representative histograms of MR on macrophages across culture conditions shown in (C, top). (D, top) Mean fluorescence intensity (MFI) of Arg1 macrophages cultured with TCM generated from tumors from untreated or TGFβ-vaccinated mice. (D, bottom) Representative histograms of Arg1 on macrophages across culture conditions shown in (D, top). (E, top) MFI of PD-L1 macrophages cultured with TCM generated from tumors from untreated or TGFβ-vaccinated mice. (E, bottom) Representative histograms of PD-L1 on macrophages across culture conditions shown in (E, top). Bar plots are presented as mean±SD. Dots represent data derived from TCM generated from pooled tumors (n=2 per group). Arg1, arginase-1; IL, interleukin; PD-L1, programmed death-ligand 1; TGFβ, transforming growth factor-β.

Article Snippet: The following antibodies/dies (purchased from BioLegend unless otherwise stated) were used for flow cytometry: CD45-PE-Cy7, CD31-FITC, FAP-biotin (R&D system), streptavidin-APC, CD90-BV605, PDPN-APC, Ly6C-AF700, CD26-PerCP-Cy5.5 (eBioscience), αSMA-Cy3 (Sigma), CD11b-PE-Cy7, CD11b-Pacific Blue, F4/80-APC, F4/80-FITC, MR-PE, MR-PE-Cy7, Ly6C-PerCP-Cy5.5, Ly6G-APC-Cy7, Arg1-PE (R&D system), PDL1-APC (BD Biosciences), CD45-FITC, CD3-AF700, CD4-BV421, CD8-BV605 (BD Biosciences), CD25-PE-Cy7, FoxP3-APC and carboxyfluorescein succinimidyl ester (CFSE, Sigma Aldrich).

Techniques: Labeling, Generated, Flow Cytometry, Staining, Derivative Assay, Cell Culture, Fluorescence

Journal: Molecular Imaging and Biology

Article Title: 124 I-Iodo-DPA-713 Positron Emission Tomography in a Hamster Model of SARS-CoV-2 Infection

doi: 10.1007/s11307-021-01638-5

Figure Lengend Snippet: Iba-1 staining and flow cytometry. a Immunohistochemistry demonstrates clusters of Iba-positive cells within pneumonic tissues. b Flow cytometry demonstrates a higher percentage of myeloid cells and CD11b + cells (macrophages, phagocytes) in males ( n = 5), than females ( n = 5). Data represented as median ± interquartile range.

Article Snippet: A single-cell suspension was stained with Zombie Aqua Fixable Viability Kit (Biolegend), resuspended in FACS buffer (1% bovine serum albumin in saline), and incubated in block buffer prior to surface staining with anti-CD11b antibody (Novus Biologicals, #NB110-89474PECY7, polyclonal) for macrophages, monocytes, and granulocytes.

Techniques: Staining, Flow Cytometry, Immunohistochemistry