Journal: Journal of the American Heart Association

Article Title: Transplantation of Allogeneic Pericytes Improves Myocardial Vascularization and Reduces Interstitial Fibrosis in a Swine Model of Reperfused Acute Myocardial Infarction

doi: 10.1161/jaha.117.006727

Figure Lengend Snippet: Figure 6. Flow cytometry analyses of APCs isolated from human and swine saphenous veins. A and B, Representative flow cytometry gating procedure of hAPC line #1 and sAPC line #1 at P5. Total cell populations and the single cells (singlets) were gated according to FSC-A vs SSC-A and FSC-A vs FSC-H parameters (i and ii). Viable cells were distinguished from dead cells using Fixable Viability Dye eFluor780 (iii) and further gated for selected antigens (iv through xi and iv through xiii). Pericyte, mesenchymal, endothelial, and hematopoietic markers were studied. The FMO control was used in the assessment and gating of CD146+ and PDGFRb+ cells, because of the use of multiple fluorochromes (vii through ix and viii through x). The same approach was used when studying the expression of CD45 and CD11b on sAPCs to exclude hematopoietic cell contamination in the cell culture system (xi through xiii). Data were acquired using FACSCantoII (BD Biosciences) or Novocyte 3000 flow cytometer (ACEA Biosciences, San Diego, CA, USA) and analyzed using the FlowJo v10.3 software. C and D, Flow cytometry histograms for each surface marker in representative hAPC (C) and sAPC lines (D). Negative control staining profiles are shown by the red histograms, whereas specific antibody staining profiles are shown by light blue histograms. Bar graphs show the meanSEM values of 3 hAPC and sAPC lines. E and F, Gating and histograms of fresh isolated swine PB-MNCs and swine PAEC line #1 at P5 used as positive control for the staining of hematopoietic and endothelial markers, respectively. In both cell lines, the negative control staining profile is shown by full red histogram, while the positive staining profile is shown by full light-blue histogram. FSC-A indicates Forward Scatter Area; FSC-H, Forward Scatter Height; FMO, fluorescence minus 1; hAPCs, human adventitial pericytes; PB-MNCs, Peripheral blood mononuclear cells; PDGFRb, platelet-derived growth factor receptor-b; sAPCs, swine adventitial pericytes.

Article Snippet: Antibodies used in flow cytometry studies on sAPCs Marker Permeabilization Reactivity Primary antibody source dilution Fluorophores CD90 No Swine eBioscience, 1:20 Pe-Cy7 CD44 No Swine eBioscience, 1:20 APC CD105 No Swine LifeSpan, 1:5 PE CD45 No Swine Serotec, 1:25 FITC CD11b No Human/Swine eBioscience, 1:20 Pe-Cy7 CD31 No Swine Serotec, 1:80 PE CD146 No Swine Serotec, 1:60 FITC PDGFR No Human Biolegend, 1:25 PE Dye eFluor 780 No eBioscience, 1:1000 APC-Cy7 Cells (1x106, N=3 biological replicates) were washed in 1X DPBS (Life Technologies) and treated with 10X trypsin/EDTA (Life Technologies).

Techniques: Flow Cytometry, Isolation, Cytometry, Control, Expressing, Cell Culture, Software, Marker, Negative Control, Staining, Positive Control, Derivative Assay

Journal: Journal of hepatology

Article Title: Adipose tissue macrophage dysfunction is associated with a breach of vascular integrity in NASH.

doi: 10.1016/j.jhep.2023.10.039

Figure Lengend Snippet: Fig. 1. Single-cell map of human VAT macrophages. (A) Workflow of the experimental pipeline. Human visceral adipose tissue was collected from patients undergoing cholecystectomy and bariatric surgery, NAFLD-stratified based on liver histology, and the stromal vascular fraction enzymatically isolated. The cells were sorted based on CD45, CD11b, and HLA-DR positivity and analyzed using 10X Genomics Chromium droplet single-cell RNA sequencing. The cells were then clustered based on marker genes and downstream analysis was performed. (B) Annotated UMAP map of 35,405 myeloid cells from the VAT of lean (n = 3) and obese patients with normal liver histology (n = 4), NAFL (n = 4) and NASH (n = 5), showing ResVAMs, MMacs, TransMacs, preInfMacs, Mon, ncMon and cDCs. (C) Heatmap showing the top 20 marker genes for the myeloid populations. Indicated are individual cluster markers and the total number of genes characterized. The expression is indicated by color saturation, the cluster and disease identity are marked by individual coloring. (D) Violin plots showing the expression of selected marker genes in myeloid cells,

Article Snippet: The cells were sorted based on CD45, CD11b, and HLA-DR positivity and analyzed using 10X Genomics Chromium droplet single-cell RNA sequencing.

Techniques: Isolation, RNA Sequencing, Marker, Expressing

Journal: PLoS Pathogens

Article Title: IL-33 receptor ST2 regulates the cognitive impairments associated with experimental cerebral malaria

doi: 10.1371/journal.ppat.1006322

Figure Lengend Snippet: (A-B) Mixed glial cell cultures from newborn WT (A) and ST2 -/- (B) mice were stimulated with rmIL-33 (10 or 100ng/mL) or LPS (10μg/mL), as a positive control for 24 hrs, or unstimulated, then fixed and used for immunofluorescence staining of IL-1β with microglia marker CD11b. Arrows indicate colocalization. These images are representative of 3 independent experiments. Scale bar 100μm. (C) Cell percentages of CD11b + IL-1β + cells from A and B. These results are expressed as mean ± SEM, n = 3 mice per group in 2 independent experiments with 5 fields analyzed per slide. Kruskal-Wallis was applied, followed by Dunn’s comparison test (*p≤0 . 05 , **p≤0 . 01) .

Article Snippet: The cells were incubated overnight with different primary antibodies raised in rat anti-CD11b (Bio-Rad MCA711G; 1:200), goat anti-IL-1β (Sigma I3767; 1:500), or rabbit anti-GFAP, rabbit anti-OLIG2, goat anti-IL-33 as above.

Techniques: Positive Control, Immunofluorescence, Staining, Marker

Journal: PLoS ONE

Article Title: Identification of Myeloid Derived Suppressor Cells in Dogs with Naturally Occurring Cancer

doi: 10.1371/journal.pone.0033274

Figure Lengend Snippet: PBMCs from healthy dogs and dogs with cancer were stained for the myeloid marker CD11b, monocytic marker CD14 and MHC II. (A) Representative flow cytometric analysis of forward and side scatter and gated CD11b + CD14 − MHCII − cells from dogs with advanced or metastatic tumors compared to dogs with early stage non-metastatic tumors and healthy control dogs. Plots are representative of dog with advanced metastatic hemangiosarcoma (top), early stage bladder transitional cell carcinoma (middle) and a healthy dog. (B) FACS sorted CD11b + CD14 − MHCII − cells were stained with diff-quick for cell morphology evaluation. A representative example of polymorphonuclear granulocyte morphology of CD11b + CD14 − MHCII − cells is shown at 63× magnification.

Article Snippet: Cells were first labeled using indirect staining with 0.1 μg of unconjugated mouse anti-dog CD11b antibody (clone CA16.3E10, AbD Serotec) or IgG1 isotype control (AbD Serotec) and 0.5 μg of PE-conjugated goat F(ab′)2 anti-mouse IgG (Abcam) secondary antibody at 4°C for 30 min in a dark room.

Techniques: Staining, Marker, Control, Diff-Quik

Journal: PLoS ONE

Article Title: Identification of Myeloid Derived Suppressor Cells in Dogs with Naturally Occurring Cancer

doi: 10.1371/journal.pone.0033274

Figure Lengend Snippet: (A) Analysis of average CD11 + CD14 − MHCII − population frequency in dogs with advanced stage or metastatic tumors (n = 30) compared with early stage non-metastatic tumors (n = 15) and control dogs (n = 18). There was a significantly higher percentage of CD11b + CD14 − MHCII − cells in dogs with advanced cancer versus early stage non-metastatic tumors and healthy dogs (36.04% vs. 9.40% and10.24%, respectively. B) Average CD11b + CD14 − MHCII − population frequency in the major cancer subtypes: advanced stage or metastatic sarcomas (n = 18), early stage non-metastatic sarcomas (n = 6), advanced stage or metastatic carcinomas (n = 7) early stage non-metastatic carcinomas (n = 7), advanced stage or metastatic mast cell tumors (n = 5) and early stage non-metastatic mast cell tumors (n = 2) compared with control dogs (n = 18). Significantly elevated percentages were detected in all advanced tumors subtypes relative to early stage tumors and healthy dogs. Percentages of CD11b + CD14 + MHCII − cells were not significant between groups (* indicates P<0.001). Mean ± SEM are shown.

Article Snippet: Cells were first labeled using indirect staining with 0.1 μg of unconjugated mouse anti-dog CD11b antibody (clone CA16.3E10, AbD Serotec) or IgG1 isotype control (AbD Serotec) and 0.5 μg of PE-conjugated goat F(ab′)2 anti-mouse IgG (Abcam) secondary antibody at 4°C for 30 min in a dark room.

Techniques: Control

Journal: PLoS ONE

Article Title: Identification of Myeloid Derived Suppressor Cells in Dogs with Naturally Occurring Cancer

doi: 10.1371/journal.pone.0033274

Figure Lengend Snippet: CD11b + CD14 − MHCII − cells were sorted from peripheral blood sample of dogs with cancer and then co-cultured with autologous PBMCs (A, B) or healthy dog PBMCs (C) in the presence of mitogen for 72 hs. Representative examples from a total of eight dogs are shown. The graphs represent proliferative responses after addition of CD11b + CD14 − MHCII − isolated from a single dog with squamous cell carcinoma (3A), prostatic adenocarcinoma (3B) and osteosarcoma (3C). Non-stimulated PBMCs were used as negative control and PBMCs stimulated in absence of CD11b + CD14 − MHCII − cells were used as positive control for proliferation. PBMCs were also co-incubated with PMNs, to control for presence of additional cells (3C, 3D). Proliferative responses were measured by 3 H-thymidine incorporation. CPM, counts per minute. Amount of IFN-γ secretion in the co-culture was determined using canine specific IFN-γ ELISA assay (3D). All experiments were performed in triplicate. Mean ± SEM are shown.

Article Snippet: Cells were first labeled using indirect staining with 0.1 μg of unconjugated mouse anti-dog CD11b antibody (clone CA16.3E10, AbD Serotec) or IgG1 isotype control (AbD Serotec) and 0.5 μg of PE-conjugated goat F(ab′)2 anti-mouse IgG (Abcam) secondary antibody at 4°C for 30 min in a dark room.

Techniques: Cell Culture, Isolation, Negative Control, Positive Control, Incubation, Control, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Identification of Myeloid Derived Suppressor Cells in Dogs with Naturally Occurring Cancer

doi: 10.1371/journal.pone.0033274

Figure Lengend Snippet: Facs sorted CD11b + CD14 − MHCII − cells isolated from a dog with osteosarcoma or healthy PBMCs were co-incubated with mitogen-stimulated CD4 + and CD8 + T cells isolated from a healthy dog for 72 hs. No stimulated cells were used as negative control. Proliferative responses were measured by 3 H-thymidine incorporation from experiments performed in triplicate. CPM, counts per minute. Mean ± SEM are shown.

Article Snippet: Cells were first labeled using indirect staining with 0.1 μg of unconjugated mouse anti-dog CD11b antibody (clone CA16.3E10, AbD Serotec) or IgG1 isotype control (AbD Serotec) and 0.5 μg of PE-conjugated goat F(ab′)2 anti-mouse IgG (Abcam) secondary antibody at 4°C for 30 min in a dark room.

Techniques: Isolation, Incubation, Negative Control

Journal: PLoS ONE

Article Title: Identification of Myeloid Derived Suppressor Cells in Dogs with Naturally Occurring Cancer

doi: 10.1371/journal.pone.0033274

Figure Lengend Snippet: RT-PCR analysis of FACS purified CD11b + CD14 − MHCII − cells detected expression of ARG1 and iNOS2, as well TGF-β and IL-10 immunosuppressive cytokines. ARG-1 expression was not detected in normal PMNs. CD11b + CD14 − MHCII − cells were isolated from the peripheral blood of a dog with osteosarcoma and PMNs were isolated from a healthy dog. NRT, RNA template in the absence of reverse transcriptase. are representative three experiments.

Article Snippet: Cells were first labeled using indirect staining with 0.1 μg of unconjugated mouse anti-dog CD11b antibody (clone CA16.3E10, AbD Serotec) or IgG1 isotype control (AbD Serotec) and 0.5 μg of PE-conjugated goat F(ab′)2 anti-mouse IgG (Abcam) secondary antibody at 4°C for 30 min in a dark room.

Techniques: Reverse Transcription Polymerase Chain Reaction, Purification, Expressing, Isolation, Reverse Transcription