anti-cd33 pe-cy7  (Thermo Fisher)

Journal: Frontiers in Immunology

Article Title: The immune cell landscape of glioblastoma patients highlights a myeloid-enriched and immune suppressed microenvironment compared to metastatic brain tumors

doi: 10.3389/fimmu.2023.1236824

Figure Lengend Snippet: Analysis of the myeloid infiltrate in the TME of BrM and GBM. (A) Schematic representation of the localization of primary BrM tumors analyzed in the study. In the upper left, a representative surgical view under white and blue light after 5-ALA administration to a BrM patient. Figure created with biorender.com. (B) Venn diagram of the samples included in the study. Venn diagram showing BrM (upper part) and GBM (lower part) blood and tumor samples included in the analysis. Overlapping numbers in the graphs refer to the numbers of patients for which matched tumor and blood samples were obtained. (C) Representative flow cytometry gating strategy for the identification of the different myeloid subsets in tumor samples. After morphological evaluation and the exclusion of doublets and dead cells, leukocytes were identified on the basis of their CD45 expression. Macrophages and PMN were further discriminated in the CD45 + gate based on their differential CD33 expression, with PMN identified as CD33 dim cells and macrophages as CD33 high cells. Finally, BMDM and MG were further differentiated in the CD33 high subset either based on the combined expression of CD49d and HLA-DR or CD33 and HLA-DR, with BMDM distinguished as CD49d + /HLA-DR + or CD33 high /HLA-DR + and MG as CD49d - /HLA-DR + or CD33 high /HLA-DR dim , respectively. (D) Analysis of the frequency of the leukocyte subsets in the TME of BrM and GBM. CD45 + cells were calculated among live cells, while CD33 high , BMDM, MG and PMN were gated among CD45 + leukocytes. Violin plots show the frequency distribution and median of tumor-infiltrating leukocytes in the whole BrM (blue plots) and GBM (red plots) (n = 14 for BrM, except for PMN [n = 13]; n = 18 for GBM). Comparison by Mann-Whitney test. * <.05; ** <.01; *** ≤.001. (E) Analysis of the leukocyte infiltrate in the TME of BrM according to their primary tumor site. Bars show the mean ± standard error (SE) of the frequency of tumor-infiltrating leukocytes in BrM from breast cancer (pink bars, n = 3), melanoma (grey bars, n = 5), lung cancer (white bars, n = 3) and GBM (red bars, n = 18). Each black dot represents a sample. Comparison by t-test. * <.05; ** <.01; *** ≤.001.

Article Snippet: To this end, single-cell suspensions obtained from freshly resected tumors were stained with LIVE/DEAD™ Fixable Aqua (Life Technologies, Thermo Fisher Scientific), anti-CD45 BV421 (BD Biosciences), anti-CD33 PE-Cy7 (eBioscience, Thermo Fisher Scientific) or anti-CD33 APC (BD Biosciences), anti-HLA-DR APC (BD Biosciences), anti-CD49d PE (BioLegend, San Diego, CA, USA), anti-CD3 PE-Cy7 (Beckman Coulter, Indianapolis, Indiana, USA) or anti-CD3 APC-H7 (BD Biosciences), anti-CD8 APC-H7 (BD Biosciences), anti-CD4 BV785 (BioLegend), anti-lymphocyte-activation gene 3 (LAG-3) FITC (AdipoGen, San Diego, CA, USA), anti-programmed cell death protein 1 (PD-1) PE (Miltenyi Biotec) and anti-T cell immunoreceptor with Ig and ITIM domains (Tigit) PE-Cy7 (BioLegend).

Techniques: Flow Cytometry, Expressing, Comparison, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: The immune cell landscape of glioblastoma patients highlights a myeloid-enriched and immune suppressed microenvironment compared to metastatic brain tumors

doi: 10.3389/fimmu.2023.1236824

Figure Lengend Snippet: Immune suppressive activity of macrophages in the TME of a lung BrM. CD33 high cells were isolated from a single-cell suspension of a lung BrM using anti-CD33 immunomagnetic beads and cultured for four days with αCD3 and αCD28 mAb-stimulated PBMC in a 1:1 ratio. (A) Dot plots show the proliferation of unstimulated T cells (left), of αCD3/αCD28 mAb-stimulated T cells (middle) and of T cells cultured in the presence of CD33 high cells (right). Green histograms below represent the model of the proliferation of T cells in the corresponding conditions, with each peak corresponding to a proliferation generation. (B) On the left, CellTrace histograms of unstimulated T cells alone (black plot), αCD3/αCD28 mAb-stimulated T cells (green plot) and T cells in the presence of sorted CD33 high cells (orange plot). On the right, the bars represent the value of quantitative T cell proliferation normalized assuming the proliferation of T cells alone (green bar) to be 100%.

Article Snippet: To this end, single-cell suspensions obtained from freshly resected tumors were stained with LIVE/DEAD™ Fixable Aqua (Life Technologies, Thermo Fisher Scientific), anti-CD45 BV421 (BD Biosciences), anti-CD33 PE-Cy7 (eBioscience, Thermo Fisher Scientific) or anti-CD33 APC (BD Biosciences), anti-HLA-DR APC (BD Biosciences), anti-CD49d PE (BioLegend, San Diego, CA, USA), anti-CD3 PE-Cy7 (Beckman Coulter, Indianapolis, Indiana, USA) or anti-CD3 APC-H7 (BD Biosciences), anti-CD8 APC-H7 (BD Biosciences), anti-CD4 BV785 (BioLegend), anti-lymphocyte-activation gene 3 (LAG-3) FITC (AdipoGen, San Diego, CA, USA), anti-programmed cell death protein 1 (PD-1) PE (Miltenyi Biotec) and anti-T cell immunoreceptor with Ig and ITIM domains (Tigit) PE-Cy7 (BioLegend).

Techniques: Activity Assay, Isolation, Suspension, Cell Culture

Journal: Cell Stem Cell

Article Title: Controlling genetic heterogeneity in gene-edited hematopoietic stem cells by single-cell expansion

doi: 10.1016/j.stem.2023.06.002

Figure Lengend Snippet:

Article Snippet: anti-human CD33-PE/Cy7 (WM53) , Thermo Fisher Scientific , Cat#25-0338-42; RRID: AB_1907380.

Techniques: Recombinant, Sequencing, Software, CRISPR

Journal: Oncology Research

Article Title: Chondroitin Sulfate Proteoglycan-4 (CSPG4)-Specific Monoclonal Antibody 225.28 in Detection of Acute Myeloid Leukemia Blasts

doi: 10.3727/096504014X14174484758503

Figure Lengend Snippet: CSPG4 expression on acute leukemia cell lines and the gating strategy used for CSPG4 detection on myeloblasts in the peripheral blood of AML patients. (a) mAb 225.28 (or isotype control IgG2a Ab) was used to demonstrate expression of CSPG4 on the surface of THP-1 and ML-2 cells maintained in culture. (b) Abs specific for CD45, CD117, CD33, or CD34 were used to identify myeloblasts. Cells were individually stained with each antibody and used to set appropriate voltage and gain for each fluorochrome against its isotype. For analysis of patient samples, mAb 225.28 was labeled with FITC fluorochrome and used for direct staining of cells prior to flow cytometry.

Article Snippet: Mouse anti-human CD33-PE-Cy7 (IgG1) and mouse anti-human CD45-PerCP-Cy5.5 (IgG1) were purchased from eBioscience (San Diego, CA, USA).

Techniques: Expressing, Staining, Labeling, Flow Cytometry

Journal: Oncology Research

Article Title: Chondroitin Sulfate Proteoglycan-4 (CSPG4)-Specific Monoclonal Antibody 225.28 in Detection of Acute Myeloid Leukemia Blasts

doi: 10.3727/096504014X14174484758503

Figure Lengend Snippet: Cytogenetic, Molecular Profile, and CSPG4 Expression Detected by mAb 225.28 in Patients With AML

Article Snippet: Mouse anti-human CD33-PE-Cy7 (IgG1) and mouse anti-human CD45-PerCP-Cy5.5 (IgG1) were purchased from eBioscience (San Diego, CA, USA).

Techniques: Expressing

Journal: bioRxiv

Article Title: Loss of FBXO11 function establishes a stem cell program in acute myeloid leukemia through dysregulation of the mitochondrial protease LONP1

doi: 10.1101/2022.09.10.507366

Figure Lengend Snippet: a , Schematic outlining the experimental design for long-term culture and xenotransplant of CD34 + HSPC. b , FBXO11 knockdown maintains CD34 + cells in long-term culture. Percentages (left panel) and total live cell counts (right panel) of CD34 + cells were measured on day 40 of culture ( n = 9 (EV), n = 3 (F, A, FA, K, FK), n = 7 (KA), n = 12 (FKA) independent replicates). c , Kaplan-Meier curve for mice transplanted with cells harvested in (b) ( P = 0.0028 for EV vs FKA, and P = 0.0002 for KA vs FKA (Mantel-Cox test)). d , Bone marrow engraftment of transduced CD34 + cells harvested in (b) (EV, empty vector + control shRNA ( n = 13); KA, KRAS G12D + AML1-ETO ( n = 9); and FKA, sh FBXO11 + KRAS G12D + AML1-ETO ( n = 12) mice) showing CD45 + cell chimerism levels in the non-injected bone (NI) or injected bone (I) ( P = 0.0033 for EV vs FKA, P = 0.018 for KA vs FKA (Sidak’s multiple comparisons test)). e , Giemsa-stained bone marrow smear from mice at endpoint. f , Human CD45 + hematopoietic cell engraftment in the spleen at endpoint as percentage of total viable spleen cells ( n = 5 (EV, 3 not detected (ND)), n = 9 (KA, 1 ND), n = 9 (FKA) mice). g , Human CD45 + hematopoietic cell engraftment in the bone marrow measured at endpoint. ( n = 13 (EV, 7 ND), n = 9 (KA), n = 12 (FKA) mice). h , Percentage of lymphoid (CD3 + , CD19 + ), myeloid (CD33 + ), and other (CD3 - CD19 - CD33 - ) cells within the viable human CD45 + cell population was measured in bone marrow cells at endpoint. ( n = 5 (EV), n = 5 (KA), n = 5 (FKA) mice). i , Total CD34 + HSPC numbers in the bone marrow were quantified at endpoint. ( n = 13 (EV, 13 ND), n = 9 (KA, 8 ND), n = 12 (FKA) mice). j , Hematopoietic cell engraftment in secondary transplants was assessed at weeks 8, 22 and 24. The table on the right lists the fraction of mice in each condition with detectable engraftment at each time point ( P values represent Fisher’s exact tests). All P values represent two-tailed t- tests unless otherwise specified, and error bars represent s.d.

Article Snippet: For transplant analysis of ex vivo cultured cells, bone marrow aspirates were performed every 6 weeks post-transplant and human chimerism was tracked by flow cytometry analysis (using a BD LSR Fortessa analyzer or BD FACSymphony) and analyzed with the following: APC-anti-CD45 2D1(1:50; Invitrogen 17-9459-42), APC/Cy7 anti-CD45 HI30 (1:50; BioLegend 304014), AF700-anti-CD34 (1:50; BD biosciences 561440), PE/Cy7-anti-CD33 (1:100; Thermo fisher 25-0338-42), PE/Cy7-anti-CD15 (1:100; BD biosciences 560827), BV650-anti-CD19 (1:100; BD biosciences 560827), SB600-anti-CD3(1:50; Invitrogen 83-0037-42), DAPI (1 μg/mL final concentration, Sigma-Aldrich D9542).

Techniques: Plasmid Preparation, shRNA, Injection, Staining, Two Tailed Test