membrane based antibody array  (R&D Systems)

Journal: Science Advances

Article Title: Leukemia-on-a-chip: Dissecting the chemoresistance mechanisms in B cell acute lymphoblastic leukemia bone marrow niche

doi: 10.1126/sciadv.aba5536

Figure Lengend Snippet: ( A ) Schematic demonstrating the design of leukemia-on-a-chip that consists of three functional regions. ( B ) Whole scan of the resultant leukemic BM niche system, where the enlarged area shows the colocalization of murine B-ALL cells within the perivascular niche. The bottom insert shows colocalization in the endosteal niche. ( C ) Hematoxylin and eosin (H&E) staining image of the in vivo murine leukemic BM niche, with the enlarged area showing the colocalization of B-ALL cells within the perivascular and endosteal niches. ( D ) The chemoresistance was compared between the engineered human BM niches of ETV6-RUNX1 + REH and Ph + SUP-B15 B-ALL. Each drug concentration had three or more experimental replicates. ( E ) The cytokine profiles from two B-ALL blasts with and without niche cells were quantified using membrane-based enzyme-linked immunosorbent assay (ELISA) analysis. MCP-1, monocyte chemoattractant protein-1; MIG, monokine induced by gamma interferon. ( F ) Quantification of nuclear (Nuc)/cytoplasmic (Cyto) ratio of NF-κB in REH and SUP B-ALL within their respective niche models. The ratios for REH and SUP were manually measured from three experimental replicates ( n > 150). ( G ) Percentage of Ki67 + B-ALL cells, corresponding to (F). Data were collected from three experimental replicates. Unpaired t test (** P < 0.01, Mann-Whitney test). GCSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; GRO, growth-regulated oncogene; IL, interleukin; IFN-γ, interferon-γ; TGFβ1, transforming growth factor–β1; TNFα, tumor necrosis factor–α; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Cytokine secretion profiles of niche cells were examined by using Mouse Cytokine Antibody Array membrane-based ELISA kit (AAM-CYT-3, RayBiotech) or Human Cytokine Antibody Array membrane-based ELISA kit (AAH-CYT-1, RayBiotech), according to the manufacturer’s protocols.

Techniques: Functional Assay, Staining, In Vivo, Concentration Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Science Advances

Article Title: Leukemia-on-a-chip: Dissecting the chemoresistance mechanisms in B cell acute lymphoblastic leukemia bone marrow niche

doi: 10.1126/sciadv.aba5536

Figure Lengend Snippet: ( A ) Regional CXCR4 distribution on murine B-ALL cells cultured with or without niche cells. ( B ) Quantified result corresponding to (A). ( C ) Membrane-based ELISA analysis of CXCL12 expression level of niche cells (ECs and MSCs). ( D ) Quantified result corresponding to (C). ( E ) Representative image showing B-ALL cells colocalized with niche cells via VCAM-1/VLA-4 signaling. ( F ) Representative images showing nuclear translocation of NF-κB in B-ALL cells. ( G ) NF-κB activation in B-ALL cells with or without niche cells and under treatments with CXCR4 inhibitor, AMD3100 (AMD), and VLA-4 inhibitor, BIO. The ratios were manually quantified from three experimental replicates ( n > 200). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. ( H ) B-ALL cell viability cultured with or without niche cells. ( I ) Quantification of NF-κB activation in human NALM-6, 697, RS(4;11), and UOCB1 blasts within their leukemia niches. The ratios were manually measured from three experimental replicates ( N > 150). ( J ) Quantification of NF-κB activation in patient-derived B-ALL blasts within engineered leukemia niches. The ratios for various B-ALL blasts were manually measured from three technical replicates ( N > 20). Unpaired t test (* P < 0.05 and ** P < 0.01, Mann-Whitney test).

Article Snippet: Cytokine secretion profiles of niche cells were examined by using Mouse Cytokine Antibody Array membrane-based ELISA kit (AAM-CYT-3, RayBiotech) or Human Cytokine Antibody Array membrane-based ELISA kit (AAH-CYT-1, RayBiotech), according to the manufacturer’s protocols.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Translocation Assay, Activation Assay, Derivative Assay, MANN-WHITNEY

Journal: Science Advances

Article Title: Leukemia-on-a-chip: Dissecting the chemoresistance mechanisms in B cell acute lymphoblastic leukemia bone marrow niche

doi: 10.1126/sciadv.aba5536

Figure Lengend Snippet: ( A ) Membrane-based ELISA analysis of CXCL12 secretion of ECs (top left) and MSCs (top right). Western blotting of CXCL12 expression of ECs (bottom left) and MSCs (bottom right). ( B ) VCAM-1 expression of ECs and the quantification result ( n > 200). ( C ) OPN expression of 2D cultured MSCs and the quantification result ( N > 200). ( D ) The correlation between distance of B-ALL to MSCs and dye-retaining ability of B-ALL ( n > 120). B-ALL with lower DiD intensity was excluded as indicated by the dashed line. ( E and F ) The flow cytometric images showing DiD dye retained in B-ALL, i.e., low (Lo), middle (Mid), and high (Hi) intensity. FSC-A, forward scatter area. ( G ) The representative images showing p21 expression in B-ALL located in the perivascular and endosteal niches ( N = 54). ( H ) B-ALL viability in the two niches treated with different drugs. ( I ) NF-κB activation in REH after coculture with hematopoietic cells. ( J ) REH viability cultured with hematopoietic cells after 48-hour treatment of 20 nM VCR. ( K ) Representative images of CD34 + HSPC within (REH) or without (Control) the leukemia niche at days 1 and 9. ( L ) Quantified number of CD34 + cells. Unpaired t test (* P < 0.05 and ** P < 0.01, Mann-Whitney test). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; a.u., arbitrary units.

Article Snippet: Cytokine secretion profiles of niche cells were examined by using Mouse Cytokine Antibody Array membrane-based ELISA kit (AAM-CYT-3, RayBiotech) or Human Cytokine Antibody Array membrane-based ELISA kit (AAH-CYT-1, RayBiotech), according to the manufacturer’s protocols.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Cell Culture, Activation Assay, MANN-WHITNEY