cd34 + hnep  (AllCells LLC)

Journal: Cell reports

Article Title: Identification of an Early Unipotent Neutrophil Progenitor with Pro-Tumoral Activity in Mouse and Human Bone Marrow

doi: 10.1016/j.celrep.2018.07.097

Figure Lengend Snippet: (A) Flow cytometry analysis of healthy human BM uncovers a heterogeneous Lin−CD66b+CD117+ hNeP fraction. Dump antibody cocktail contains antibodies against markers that are expressed by: HSC (CD90 (Thy1)), lymphocytes and their progenitors (CD3, CD19, CD56, CD161, CD7, CD127 (IL-7Rα)), Erythrocytes and their progenitors (CD41, CD235a (Glycophorin A)), Eosinophils/Basophils and their progenitors (Siglec 8, FcεRIα, , CD125 (IL-5Rα)), CMP/GMP and monocyte progenitors (CD123 (IL-3Rα)), DCs and macrophages (CD11c, CD169). (B) ScRNA-Seq analysis of Lin−CD66b+CD117+ hNeP cells reveals two major subpopulations, Subset A and Subset B. 20,000 hNeP cells were FACS sorted from healthy human BM for scRNA-Seq. Heatmap shows top 40 differentially expressed genes in each cluster. Log2 Fold Change of each gene expression is relative to the entire dataset. 2 biological triplicates, 2 technical replicates. (C) Lin−CD66b+CD117+ hNeP were divided into CD34+ subset and CD34−subset by flow cytometry. Confocal microscopy was used to detect Ki67 localization (red) within the nuclei (blue) in CD34+ subset and CD34−subset using antibodies to Ki67 and Hoechst. IgG stained cells served as negative control. Bar: 5µm.

Article Snippet: CD34 + hNeP, CD34 − hNeP, and mature neutrophils were FACS sorted from fresh human BM obtained from AllCells, Inc. (Alameda, CA).

Techniques: Flow Cytometry, Gene Expression, Confocal Microscopy, Staining, Negative Control

Journal: Cell reports

Article Title: Identification of an Early Unipotent Neutrophil Progenitor with Pro-Tumoral Activity in Mouse and Human Bone Marrow

doi: 10.1016/j.celrep.2018.07.097

Figure Lengend Snippet: (A) Scheme showing the experimental procedure. CD34+ hNeP and CD34−hNeP subsets identified in Figure 5C were sorted from healthy human BM, and adoptively transferred into NSG-M3 recipient mice. Each recipient mouse received 25,000 donor human hNeP progenitor cells. After the transfer, peripheral blood was collected from each recipient via saphenous vein for flow cytometry on Day (D) 5, 7, 14, 28 (D5, D7, D14, D28), respectively. (B) Representative plots show the appearance of monocytes (CD86+ CD66b−), neutrophils (CD86−Siglec 8−CD66b+), eosinophils (Siglec 8+), and lymphocytes (hLy+) in each recipient group at the time points indicated. hLy antibody cocktail contains CD3, CD19, and CD56. N = 10 mice for each group.

Article Snippet: CD34 + hNeP, CD34 − hNeP, and mature neutrophils were FACS sorted from fresh human BM obtained from AllCells, Inc. (Alameda, CA).

Techniques: Flow Cytometry

Journal: Cell reports

Article Title: Identification of an Early Unipotent Neutrophil Progenitor with Pro-Tumoral Activity in Mouse and Human Bone Marrow

doi: 10.1016/j.celrep.2018.07.097

Figure Lengend Snippet: (A) hNeP is increased in melanoma patient blood. hNeP frequency was detected by flow cytometry in peripheral blood collected from healthy donors (N = 5) and melanoma patients (N = 5). Error bars indicate mean ± SEM. (B) Left, scheme showing the experiment procedure. CD34+ hNeP subset, CD34−hNeP subset, and human cMoP were FACS sorted from healthy human BM. The 3 populations were adoptively transferred into NSG-M3 recipient mice. Each recipient mouse received 25,000 donor human progenitor cells. Blank control group received only PBS for adoptive transfer. The next day, 1 × 106 143B human osteosarcoma cells were SubQ injected into each recipient mouse. Right, the tumor volume in each recipient was measured at 10d post-injection. N = 5 mice in each group. Error bars indicate mean ± SEM. (C) CD34+ hNeP cells blunt T cell activation. FACS sorting strategies for CD34+ hNeP and CD34−hNeP are shown in Figure 5A and ​andCC using using flow cytometry; FACS sorting strategies for mature neutrophils is shown in Figure S9A using flow cytometry. Histogram overlay of CD69 for CD3+ T cells cultured with CD34+ hNeP, CD34−hNeP, and mature neutrophils, in the presence of anti-CD3 for 24 hours. CD3+ T cells alone cultured without anti-CD3 served as the negative control group, and CD3+ T cells alone cultured with anti-CD3, CD28 served as the positive control group for T cell activation.

Article Snippet: CD34 + hNeP, CD34 − hNeP, and mature neutrophils were FACS sorted from fresh human BM obtained from AllCells, Inc. (Alameda, CA).

Techniques: Flow Cytometry, Control, Adoptive Transfer Assay, Injection, Activation Assay, Cell Culture, Negative Control, Positive Control

Journal: Cell reports

Article Title: Identification of an Early Unipotent Neutrophil Progenitor with Pro-Tumoral Activity in Mouse and Human Bone Marrow

doi: 10.1016/j.celrep.2018.07.097

Figure Lengend Snippet: (A) Flow cytometry analysis of healthy human BM uncovers a heterogeneous Lin−CD66b+CD117+ hNeP fraction. Dump antibody cocktail contains antibodies against markers that are expressed by: HSC (CD90 (Thy1)), lymphocytes and their progenitors (CD3, CD19, CD56, CD161, CD7, CD127 (IL-7Rα)), Erythrocytes and their progenitors (CD41, CD235a (Glycophorin A)), Eosinophils/Basophils and their progenitors (Siglec 8, FcεRIα, , CD125 (IL-5Rα)), CMP/GMP and monocyte progenitors (CD123 (IL-3Rα)), DCs and macrophages (CD11c, CD169). (B) ScRNA-Seq analysis of Lin−CD66b+CD117+ hNeP cells reveals two major subpopulations, Subset A and Subset B. 20,000 hNeP cells were FACS sorted from healthy human BM for scRNA-Seq. Heatmap shows top 40 differentially expressed genes in each cluster. Log2 Fold Change of each gene expression is relative to the entire dataset. 2 biological triplicates, 2 technical replicates. (C) Lin−CD66b+CD117+ hNeP were divided into CD34+ subset and CD34−subset by flow cytometry. Confocal microscopy was used to detect Ki67 localization (red) within the nuclei (blue) in CD34+ subset and CD34−subset using antibodies to Ki67 and Hoechst. IgG stained cells served as negative control. Bar: 5µm.

Article Snippet: CD34 + hNeP, CD34 − hNeP, and mature neutrophils were FACS sorted from fresh human BM obtained from AllCells, Inc. (Alameda, CA).

Techniques: Flow Cytometry, Expressing, Confocal Microscopy, Staining, Negative Control

Journal: Cell reports

Article Title: Identification of an Early Unipotent Neutrophil Progenitor with Pro-Tumoral Activity in Mouse and Human Bone Marrow

doi: 10.1016/j.celrep.2018.07.097

Figure Lengend Snippet: (A) Scheme showing the experimental procedure. CD34+ hNeP and CD34−hNeP subsets identified in Figure 5C were sorted from healthy human BM, and adoptively transferred into NSG-M3 recipient mice. Each recipient mouse received 25,000 donor human hNeP progenitor cells. After the transfer, peripheral blood was collected from each recipient via saphenous vein for flow cytometry on Day (D) 5, 7, 14, 28 (D5, D7, D14, D28), respectively. (B) Representative plots show the appearance of monocytes (CD86+ CD66b−), neutrophils (CD86−Siglec 8−CD66b+), eosinophils (Siglec 8+), and lymphocytes (hLy+) in each recipient group at the time points indicated. hLy antibody cocktail contains CD3, CD19, and CD56. N = 10 mice for each group.

Article Snippet: CD34 + hNeP, CD34 − hNeP, and mature neutrophils were FACS sorted from fresh human BM obtained from AllCells, Inc. (Alameda, CA).

Techniques: Flow Cytometry

Journal: Cell reports

Article Title: Identification of an Early Unipotent Neutrophil Progenitor with Pro-Tumoral Activity in Mouse and Human Bone Marrow

doi: 10.1016/j.celrep.2018.07.097

Figure Lengend Snippet: (A) hNeP is increased in melanoma patient blood. hNeP frequency was detected by flow cytometry in peripheral blood collected from healthy donors (N = 5) and melanoma patients (N = 5). Error bars indicate mean ± SEM. (B) Left, scheme showing the experiment procedure. CD34+ hNeP subset, CD34−hNeP subset, and human cMoP were FACS sorted from healthy human BM. The 3 populations were adoptively transferred into NSG-M3 recipient mice. Each recipient mouse received 25,000 donor human progenitor cells. Blank control group received only PBS for adoptive transfer. The next day, 1 × 106 143B human osteosarcoma cells were SubQ injected into each recipient mouse. Right, the tumor volume in each recipient was measured at 10d post-injection. N = 5 mice in each group. Error bars indicate mean ± SEM. (C) CD34+ hNeP cells blunt T cell activation. FACS sorting strategies for CD34+ hNeP and CD34−hNeP are shown in Figure 5A and ​andCC using using flow cytometry; FACS sorting strategies for mature neutrophils is shown in Figure S9A using flow cytometry. Histogram overlay of CD69 for CD3+ T cells cultured with CD34+ hNeP, CD34−hNeP, and mature neutrophils, in the presence of anti-CD3 for 24 hours. CD3+ T cells alone cultured without anti-CD3 served as the negative control group, and CD3+ T cells alone cultured with anti-CD3, CD28 served as the positive control group for T cell activation.

Article Snippet: CD34 + hNeP, CD34 − hNeP, and mature neutrophils were FACS sorted from fresh human BM obtained from AllCells, Inc. (Alameda, CA).

Techniques: Flow Cytometry, Adoptive Transfer Assay, Injection, Activation Assay, Cell Culture, Negative Control, Positive Control