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PI3K/AKT signaling regulates CXCR4 expression in BCR-dependent DLBCL cell lines. (A) BCR-dependent DLBCL cell lines, DHL4, DHL6, LY7 and TMD8, were retrovirally transduced with pMIG-mAKT1-IRES-GFP or pMIG-IRES-GFP vector, FACS-sorted for GFP expression, treated with 1 μM R406 or vehicle for 24 h (h), and analyzed for CXCR4 expression by flow <t>cytometry.</t> (B) BCR-dependent DLBCL cell lines were treated with 1 μM R406 (red), 10 μM LY294002 (blue) or vehicle for 24 h. Thereafter, CXCR4 expression was analyzed by qRT-PCR relative to PPIA. The P -values for vehicle versus R406 treated or vehicle versus LY294002 treated were determined with a one-sided Welch t -test. *** P
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1) Product Images from "CXCR4 upregulation is an indicator of sensitivity to B-cell receptor/PI3K blockade and a potential resistance mechanism in B-cell receptor-dependent diffuse large B-cell lymphomas"

Article Title: CXCR4 upregulation is an indicator of sensitivity to B-cell receptor/PI3K blockade and a potential resistance mechanism in B-cell receptor-dependent diffuse large B-cell lymphomas

Journal: Haematologica

doi: 10.3324/haematol.2019.216218

PI3K/AKT signaling regulates CXCR4 expression in BCR-dependent DLBCL cell lines. (A) BCR-dependent DLBCL cell lines, DHL4, DHL6, LY7 and TMD8, were retrovirally transduced with pMIG-mAKT1-IRES-GFP or pMIG-IRES-GFP vector, FACS-sorted for GFP expression, treated with 1 μM R406 or vehicle for 24 h (h), and analyzed for CXCR4 expression by flow cytometry. (B) BCR-dependent DLBCL cell lines were treated with 1 μM R406 (red), 10 μM LY294002 (blue) or vehicle for 24 h. Thereafter, CXCR4 expression was analyzed by qRT-PCR relative to PPIA. The P -values for vehicle versus R406 treated or vehicle versus LY294002 treated were determined with a one-sided Welch t -test. *** P
Figure Legend Snippet: PI3K/AKT signaling regulates CXCR4 expression in BCR-dependent DLBCL cell lines. (A) BCR-dependent DLBCL cell lines, DHL4, DHL6, LY7 and TMD8, were retrovirally transduced with pMIG-mAKT1-IRES-GFP or pMIG-IRES-GFP vector, FACS-sorted for GFP expression, treated with 1 μM R406 or vehicle for 24 h (h), and analyzed for CXCR4 expression by flow cytometry. (B) BCR-dependent DLBCL cell lines were treated with 1 μM R406 (red), 10 μM LY294002 (blue) or vehicle for 24 h. Thereafter, CXCR4 expression was analyzed by qRT-PCR relative to PPIA. The P -values for vehicle versus R406 treated or vehicle versus LY294002 treated were determined with a one-sided Welch t -test. *** P

Techniques Used: Expressing, Transduction, Plasmid Preparation, FACS, Flow Cytometry, Quantitative RT-PCR

CXCR4 expression in BCR-dependent and BCR-independent DLBCL cell lines following SYK, PI3K or BTK inhibition. BCR-dependent diffuse large B-cell lymphoma (DLBCL) cell lines (DHL4, DHL6 and TMD8) and a BCR-independent DLBCL cell line (Toledo) were treated with DMSO, R406 (1 μM), GS-9973 (2 μM), LY294002 (10 μM), GDC-0941 (0.5 μM), CAL101 (2 μM), IPI145 (1 μM), PCI-32765 (0.1 μM) for 24 hours (h). Thereafter, CXCR4 expression was analyzed by qRT-PCR relative to PPIA (A) or flow cytometry (B) as in Figure 1A and Figure 1C . (A) CXCR4 transcript abundance. Fold changes in CXCR4 transcript abundance relative to DMSO are shown below each inhibitor for the four cell lines. The P -values for vehicle versus R406, GS-9973, LY294002, GDC-0941, CAL101, IPI145 and PCI-32765 treated were determined with a one-sided Welch t -test. *** P
Figure Legend Snippet: CXCR4 expression in BCR-dependent and BCR-independent DLBCL cell lines following SYK, PI3K or BTK inhibition. BCR-dependent diffuse large B-cell lymphoma (DLBCL) cell lines (DHL4, DHL6 and TMD8) and a BCR-independent DLBCL cell line (Toledo) were treated with DMSO, R406 (1 μM), GS-9973 (2 μM), LY294002 (10 μM), GDC-0941 (0.5 μM), CAL101 (2 μM), IPI145 (1 μM), PCI-32765 (0.1 μM) for 24 hours (h). Thereafter, CXCR4 expression was analyzed by qRT-PCR relative to PPIA (A) or flow cytometry (B) as in Figure 1A and Figure 1C . (A) CXCR4 transcript abundance. Fold changes in CXCR4 transcript abundance relative to DMSO are shown below each inhibitor for the four cell lines. The P -values for vehicle versus R406, GS-9973, LY294002, GDC-0941, CAL101, IPI145 and PCI-32765 treated were determined with a one-sided Welch t -test. *** P

Techniques Used: Expressing, Inhibition, Quantitative RT-PCR, Flow Cytometry

2) Product Images from "Molecular mechanisms for enhancement of stromal cell-derived factor 1–induced chemotaxis by platelet endothelial cell adhesion molecule 1 (PECAM-1)"

Article Title: Molecular mechanisms for enhancement of stromal cell-derived factor 1–induced chemotaxis by platelet endothelial cell adhesion molecule 1 (PECAM-1)

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M117.779603

PECAM-1 interacts with CXCR4 on the cell surface to reduce its internalization after SDF-1 stimulation. A , 293T cells were transfected with plasmids encoding PECAM-1-WT ( W ), PECAM-1-Mut ( M ), and 3xHA-tagged CXCR4 as indicated. Cells were stimulated with 50 ng/ml SDF-1 for 1 min or left untreated as indicated before lysis. Anti-PECAM-1 or anti-HA immunoprecipitates ( IP ) as indicated were subjected to immunoblot analysis using the indicated antibodies. Positions of PECAM-1 are indicated by asterisks. Arrows indicate positions of HA-CXCR4. Positions of molecular weight markers are also indicated. B , 32Dcl3 cells, starved of IL-3 for 4 h, were treated with or without 50 ng/m SDF-1 for 5 min as indicated. Cells were then stained with anti-mouse CXCR4 antibody conjugated with PE ( red ) and anti-mouse PECAM-1 ( mPECAM-1 ) antibody conjugated with APC ( green ) as well as with DAPI ( blue ) for nuclear staining. Representative confocal images of cells are shown. Scale bars correspond to 10 μm. C , 32Dcl3 cells overexpressing human PECAM-1-WT, starved of IL-3 for 4 h, were stained with anti-mouse CXCR4 ( m-CXCR4 ) antibody conjugated with PE ( red ) and anti-human PECAM-1 ( hPECAM-1 ) antibody conjugated with Alexa Fluor 647 ( green ) as well as with DAPI ( blue ) for nuclear staining. D , 32Dcl3 cells overexpressing human PECAM-1-WT ( WT ) or vector control cells ( Cont. ) were starved, and surface expression levels of CXCR4 were analyzed by flow cytometry. Iso. , isotype control. E , 32Dcl3 cells expressing PECAM-1 shRNA ( KD ) or luciferase shRNA as a control ( Cont. ) were analyzed as described for D. F , 32Dcl3 cells expressing PECAM-1 shRNA ( KD ) or luciferase shRNA as a control ( Cont. ) were stimulated with 100 ng/ml SDF-1 for the indicated times, and surface expression levels of CXCR4 were analyzed by flow cytometry. Changes in CXCR4 expression levels as estimated by mean fluorescence intensity after SDF-1 stimulation are plotted. G , 32Dcl3 cells overexpressing human PECAM-1-WT ( WT ) or human PECAM-1-Mut ( Mut ) as well as vector control cells ( Cont. ) were stimulated as described for F . All the data shown are representative of experiments repeated at least three times.
Figure Legend Snippet: PECAM-1 interacts with CXCR4 on the cell surface to reduce its internalization after SDF-1 stimulation. A , 293T cells were transfected with plasmids encoding PECAM-1-WT ( W ), PECAM-1-Mut ( M ), and 3xHA-tagged CXCR4 as indicated. Cells were stimulated with 50 ng/ml SDF-1 for 1 min or left untreated as indicated before lysis. Anti-PECAM-1 or anti-HA immunoprecipitates ( IP ) as indicated were subjected to immunoblot analysis using the indicated antibodies. Positions of PECAM-1 are indicated by asterisks. Arrows indicate positions of HA-CXCR4. Positions of molecular weight markers are also indicated. B , 32Dcl3 cells, starved of IL-3 for 4 h, were treated with or without 50 ng/m SDF-1 for 5 min as indicated. Cells were then stained with anti-mouse CXCR4 antibody conjugated with PE ( red ) and anti-mouse PECAM-1 ( mPECAM-1 ) antibody conjugated with APC ( green ) as well as with DAPI ( blue ) for nuclear staining. Representative confocal images of cells are shown. Scale bars correspond to 10 μm. C , 32Dcl3 cells overexpressing human PECAM-1-WT, starved of IL-3 for 4 h, were stained with anti-mouse CXCR4 ( m-CXCR4 ) antibody conjugated with PE ( red ) and anti-human PECAM-1 ( hPECAM-1 ) antibody conjugated with Alexa Fluor 647 ( green ) as well as with DAPI ( blue ) for nuclear staining. D , 32Dcl3 cells overexpressing human PECAM-1-WT ( WT ) or vector control cells ( Cont. ) were starved, and surface expression levels of CXCR4 were analyzed by flow cytometry. Iso. , isotype control. E , 32Dcl3 cells expressing PECAM-1 shRNA ( KD ) or luciferase shRNA as a control ( Cont. ) were analyzed as described for D. F , 32Dcl3 cells expressing PECAM-1 shRNA ( KD ) or luciferase shRNA as a control ( Cont. ) were stimulated with 100 ng/ml SDF-1 for the indicated times, and surface expression levels of CXCR4 were analyzed by flow cytometry. Changes in CXCR4 expression levels as estimated by mean fluorescence intensity after SDF-1 stimulation are plotted. G , 32Dcl3 cells overexpressing human PECAM-1-WT ( WT ) or human PECAM-1-Mut ( Mut ) as well as vector control cells ( Cont. ) were stimulated as described for F . All the data shown are representative of experiments repeated at least three times.

Techniques Used: Transfection, Lysis, Molecular Weight, Staining, Plasmid Preparation, Expressing, Flow Cytometry, Cytometry, shRNA, Luciferase, Fluorescence

A schematic model for the molecular mechanisms underlying the enhancing effect of PECAM-1 on CXCR4-mediated chemotactic signaling.
Figure Legend Snippet: A schematic model for the molecular mechanisms underlying the enhancing effect of PECAM-1 on CXCR4-mediated chemotactic signaling.

Techniques Used:

The S324A/S325A mutation in CXCR4 impairs endocytosis and prolongs SDF-1–induced activation of the PI3K/Akt/mTORC1 pathway and Rap1 to enhance chemotaxis. A , 32Dcl3 cells overexpressing human CXCR4-WT or human CXCR4-SA ( SA ) were cultured for 2 h with or without 500 ng/ml SDF-1 as indicated, and cell surface expression of human CXCR4 ( h-CXCR 4) was evaluated by flow cytometry. Red lines represent isotype controls. B , 32Dcl3 cells overexpressing human CXCR4 wild type ( WT ) or human CXCR4-SA ( SA ) were stimulated with 500 ng/ml SDF-1 for the indicated times, and surface expression levels of human CXCR4 were analyzed by flow cytometry. Changes in CXCR4 expression levels after SDF-1 stimulation are plotted. C , 32Dcl3 cells overexpressing CXCR4-WT ( WT ) or CXCR4-SA ( SA ) as well as vector control cells ( Cont. ) were subjected to chemotaxis assay with 50 ng/ml SDF-1. Relative chemotaxis was calculated by dividing the percentage of migrated cells by that of vector control cells. The data represent the mean of triplicates, and the asterisks indicate statistically significant ( p
Figure Legend Snippet: The S324A/S325A mutation in CXCR4 impairs endocytosis and prolongs SDF-1–induced activation of the PI3K/Akt/mTORC1 pathway and Rap1 to enhance chemotaxis. A , 32Dcl3 cells overexpressing human CXCR4-WT or human CXCR4-SA ( SA ) were cultured for 2 h with or without 500 ng/ml SDF-1 as indicated, and cell surface expression of human CXCR4 ( h-CXCR 4) was evaluated by flow cytometry. Red lines represent isotype controls. B , 32Dcl3 cells overexpressing human CXCR4 wild type ( WT ) or human CXCR4-SA ( SA ) were stimulated with 500 ng/ml SDF-1 for the indicated times, and surface expression levels of human CXCR4 were analyzed by flow cytometry. Changes in CXCR4 expression levels after SDF-1 stimulation are plotted. C , 32Dcl3 cells overexpressing CXCR4-WT ( WT ) or CXCR4-SA ( SA ) as well as vector control cells ( Cont. ) were subjected to chemotaxis assay with 50 ng/ml SDF-1. Relative chemotaxis was calculated by dividing the percentage of migrated cells by that of vector control cells. The data represent the mean of triplicates, and the asterisks indicate statistically significant ( p

Techniques Used: Mutagenesis, Activation Assay, Chemotaxis Assay, Cell Culture, Expressing, Flow Cytometry, Cytometry, Plasmid Preparation

3) Product Images from "Molecular mechanisms for enhancement of stromal cell-derived factor 1–induced chemotaxis by platelet endothelial cell adhesion molecule 1 (PECAM-1)"

Article Title: Molecular mechanisms for enhancement of stromal cell-derived factor 1–induced chemotaxis by platelet endothelial cell adhesion molecule 1 (PECAM-1)

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M117.779603

PECAM-1 interacts with CXCR4 on the cell surface to reduce its internalization after SDF-1 stimulation. A , 293T cells were transfected with plasmids encoding PECAM-1-WT ( W ), PECAM-1-Mut ( M ), and 3xHA-tagged CXCR4 as indicated. Cells were stimulated with 50 ng/ml SDF-1 for 1 min or left untreated as indicated before lysis. Anti-PECAM-1 or anti-HA immunoprecipitates ( IP ) as indicated were subjected to immunoblot analysis using the indicated antibodies. Positions of PECAM-1 are indicated by asterisks. Arrows indicate positions of HA-CXCR4. Positions of molecular weight markers are also indicated. B , 32Dcl3 cells, starved of IL-3 for 4 h, were treated with or without 50 ng/m SDF-1 for 5 min as indicated. Cells were then stained with anti-mouse CXCR4 antibody conjugated with PE ( red ) and anti-mouse PECAM-1 ( mPECAM-1 ) antibody conjugated with APC ( green ) as well as with DAPI ( blue ) for nuclear staining. Representative confocal images of cells are shown. Scale bars correspond to 10 μm. C , 32Dcl3 cells overexpressing human PECAM-1-WT, starved of IL-3 for 4 h, were stained with anti-mouse CXCR4 ( m-CXCR4 ) antibody conjugated with PE ( red ) and anti-human PECAM-1 ( hPECAM-1 ) antibody conjugated with Alexa Fluor 647 ( green ) as well as with DAPI ( blue ) for nuclear staining. D , 32Dcl3 cells overexpressing human PECAM-1-WT ( WT ) or vector control cells ( Cont. ) were starved, and surface expression levels of CXCR4 were analyzed by flow cytometry. Iso. , isotype control. E , 32Dcl3 cells expressing PECAM-1 shRNA ( KD ) or luciferase shRNA as a control ( Cont. ) were analyzed as described for D. F , 32Dcl3 cells expressing PECAM-1 shRNA ( KD ) or luciferase shRNA as a control ( Cont. ) were stimulated with 100 ng/ml SDF-1 for the indicated times, and surface expression levels of CXCR4 were analyzed by flow cytometry. Changes in CXCR4 expression levels as estimated by mean fluorescence intensity after SDF-1 stimulation are plotted. G , 32Dcl3 cells overexpressing human PECAM-1-WT ( WT ) or human PECAM-1-Mut ( Mut ) as well as vector control cells ( Cont. ) were stimulated as described for F . All the data shown are representative of experiments repeated at least three times.
Figure Legend Snippet: PECAM-1 interacts with CXCR4 on the cell surface to reduce its internalization after SDF-1 stimulation. A , 293T cells were transfected with plasmids encoding PECAM-1-WT ( W ), PECAM-1-Mut ( M ), and 3xHA-tagged CXCR4 as indicated. Cells were stimulated with 50 ng/ml SDF-1 for 1 min or left untreated as indicated before lysis. Anti-PECAM-1 or anti-HA immunoprecipitates ( IP ) as indicated were subjected to immunoblot analysis using the indicated antibodies. Positions of PECAM-1 are indicated by asterisks. Arrows indicate positions of HA-CXCR4. Positions of molecular weight markers are also indicated. B , 32Dcl3 cells, starved of IL-3 for 4 h, were treated with or without 50 ng/m SDF-1 for 5 min as indicated. Cells were then stained with anti-mouse CXCR4 antibody conjugated with PE ( red ) and anti-mouse PECAM-1 ( mPECAM-1 ) antibody conjugated with APC ( green ) as well as with DAPI ( blue ) for nuclear staining. Representative confocal images of cells are shown. Scale bars correspond to 10 μm. C , 32Dcl3 cells overexpressing human PECAM-1-WT, starved of IL-3 for 4 h, were stained with anti-mouse CXCR4 ( m-CXCR4 ) antibody conjugated with PE ( red ) and anti-human PECAM-1 ( hPECAM-1 ) antibody conjugated with Alexa Fluor 647 ( green ) as well as with DAPI ( blue ) for nuclear staining. D , 32Dcl3 cells overexpressing human PECAM-1-WT ( WT ) or vector control cells ( Cont. ) were starved, and surface expression levels of CXCR4 were analyzed by flow cytometry. Iso. , isotype control. E , 32Dcl3 cells expressing PECAM-1 shRNA ( KD ) or luciferase shRNA as a control ( Cont. ) were analyzed as described for D. F , 32Dcl3 cells expressing PECAM-1 shRNA ( KD ) or luciferase shRNA as a control ( Cont. ) were stimulated with 100 ng/ml SDF-1 for the indicated times, and surface expression levels of CXCR4 were analyzed by flow cytometry. Changes in CXCR4 expression levels as estimated by mean fluorescence intensity after SDF-1 stimulation are plotted. G , 32Dcl3 cells overexpressing human PECAM-1-WT ( WT ) or human PECAM-1-Mut ( Mut ) as well as vector control cells ( Cont. ) were stimulated as described for F . All the data shown are representative of experiments repeated at least three times.

Techniques Used: Transfection, Lysis, Molecular Weight, Staining, Plasmid Preparation, Expressing, Flow Cytometry, Cytometry, shRNA, Luciferase, Fluorescence

A schematic model for the molecular mechanisms underlying the enhancing effect of PECAM-1 on CXCR4-mediated chemotactic signaling.
Figure Legend Snippet: A schematic model for the molecular mechanisms underlying the enhancing effect of PECAM-1 on CXCR4-mediated chemotactic signaling.

Techniques Used:

The S324A/S325A mutation in CXCR4 impairs endocytosis and prolongs SDF-1–induced activation of the PI3K/Akt/mTORC1 pathway and Rap1 to enhance chemotaxis. A , 32Dcl3 cells overexpressing human CXCR4-WT or human CXCR4-SA ( SA ) were cultured for 2 h with or without 500 ng/ml SDF-1 as indicated, and cell surface expression of human CXCR4 ( h-CXCR 4) was evaluated by flow cytometry. Red lines represent isotype controls. B , 32Dcl3 cells overexpressing human CXCR4 wild type ( WT ) or human CXCR4-SA ( SA ) were stimulated with 500 ng/ml SDF-1 for the indicated times, and surface expression levels of human CXCR4 were analyzed by flow cytometry. Changes in CXCR4 expression levels after SDF-1 stimulation are plotted. C , 32Dcl3 cells overexpressing CXCR4-WT ( WT ) or CXCR4-SA ( SA ) as well as vector control cells ( Cont. ) were subjected to chemotaxis assay with 50 ng/ml SDF-1. Relative chemotaxis was calculated by dividing the percentage of migrated cells by that of vector control cells. The data represent the mean of triplicates, and the asterisks indicate statistically significant ( p
Figure Legend Snippet: The S324A/S325A mutation in CXCR4 impairs endocytosis and prolongs SDF-1–induced activation of the PI3K/Akt/mTORC1 pathway and Rap1 to enhance chemotaxis. A , 32Dcl3 cells overexpressing human CXCR4-WT or human CXCR4-SA ( SA ) were cultured for 2 h with or without 500 ng/ml SDF-1 as indicated, and cell surface expression of human CXCR4 ( h-CXCR 4) was evaluated by flow cytometry. Red lines represent isotype controls. B , 32Dcl3 cells overexpressing human CXCR4 wild type ( WT ) or human CXCR4-SA ( SA ) were stimulated with 500 ng/ml SDF-1 for the indicated times, and surface expression levels of human CXCR4 were analyzed by flow cytometry. Changes in CXCR4 expression levels after SDF-1 stimulation are plotted. C , 32Dcl3 cells overexpressing CXCR4-WT ( WT ) or CXCR4-SA ( SA ) as well as vector control cells ( Cont. ) were subjected to chemotaxis assay with 50 ng/ml SDF-1. Relative chemotaxis was calculated by dividing the percentage of migrated cells by that of vector control cells. The data represent the mean of triplicates, and the asterisks indicate statistically significant ( p

Techniques Used: Mutagenesis, Activation Assay, Chemotaxis Assay, Cell Culture, Expressing, Flow Cytometry, Cytometry, Plasmid Preparation

4) Product Images from "CXCR4 upregulation is an indicator of sensitivity to B-cell receptor/PI3K blockade and a potential resistance mechanism in B-cell receptor-dependent diffuse large B-cell lymphomas"

Article Title: CXCR4 upregulation is an indicator of sensitivity to B-cell receptor/PI3K blockade and a potential resistance mechanism in B-cell receptor-dependent diffuse large B-cell lymphomas

Journal: Haematologica

doi: 10.3324/haematol.2019.216218

PI3K/AKT signaling regulates CXCR4 expression in BCR-dependent DLBCL cell lines. (A) BCR-dependent DLBCL cell lines, DHL4, DHL6, LY7 and TMD8, were retrovirally transduced with pMIG-mAKT1-IRES-GFP or pMIG-IRES-GFP vector, FACS-sorted for GFP expression, treated with 1 μM R406 or vehicle for 24 h (h), and analyzed for CXCR4 expression by flow cytometry. (B) BCR-dependent DLBCL cell lines were treated with 1 μM R406 (red), 10 μM LY294002 (blue) or vehicle for 24 h. Thereafter, CXCR4 expression was analyzed by qRT-PCR relative to PPIA. The P -values for vehicle versus R406 treated or vehicle versus LY294002 treated were determined with a one-sided Welch t -test. *** P
Figure Legend Snippet: PI3K/AKT signaling regulates CXCR4 expression in BCR-dependent DLBCL cell lines. (A) BCR-dependent DLBCL cell lines, DHL4, DHL6, LY7 and TMD8, were retrovirally transduced with pMIG-mAKT1-IRES-GFP or pMIG-IRES-GFP vector, FACS-sorted for GFP expression, treated with 1 μM R406 or vehicle for 24 h (h), and analyzed for CXCR4 expression by flow cytometry. (B) BCR-dependent DLBCL cell lines were treated with 1 μM R406 (red), 10 μM LY294002 (blue) or vehicle for 24 h. Thereafter, CXCR4 expression was analyzed by qRT-PCR relative to PPIA. The P -values for vehicle versus R406 treated or vehicle versus LY294002 treated were determined with a one-sided Welch t -test. *** P

Techniques Used: Expressing, Transduction, Plasmid Preparation, FACS, Flow Cytometry, Quantitative RT-PCR

5) Product Images from "Recovery and Biodistribution of Ex Vivo Expanded Human Erythroblasts Injected into NOD/SCID/IL2R γnull mice"

Article Title: Recovery and Biodistribution of Ex Vivo Expanded Human Erythroblasts Injected into NOD/SCID/IL2R γnull mice

Journal: Stem Cells International

doi: 10.4061/2011/673752

Ex vivo expanded extGLuc + EBs express high level of eGFP by FACS analyses. Maturation profile and transfection efficiency of nontransfected (as control, top panels) and transfected (bottom panels) EBs at day 9 of HEMA culture identified on the basis of CD36/CD235a and eGFP expression (on the left) and on the basis of morphology (May-Grunwald staining, on the right), magnification 40x. The transfected EBs express great levels of eGFP. The transfection did not alter the morphology of the cells but slightly reduced the expression of the CD235a probably due to interference with the GFP signal. At day 9, in transfected and control cultures the fold increase was 48-fold and 325-fold, respectively, and the viability was 98% in both cases.
Figure Legend Snippet: Ex vivo expanded extGLuc + EBs express high level of eGFP by FACS analyses. Maturation profile and transfection efficiency of nontransfected (as control, top panels) and transfected (bottom panels) EBs at day 9 of HEMA culture identified on the basis of CD36/CD235a and eGFP expression (on the left) and on the basis of morphology (May-Grunwald staining, on the right), magnification 40x. The transfected EBs express great levels of eGFP. The transfection did not alter the morphology of the cells but slightly reduced the expression of the CD235a probably due to interference with the GFP signal. At day 9, in transfected and control cultures the fold increase was 48-fold and 325-fold, respectively, and the viability was 98% in both cases.

Techniques Used: Ex Vivo, FACS, Transfection, Expressing, Staining

Adhesion receptor profiling during in vitro maturation of EBs expanded from AB (a) and CB (b). Three color flow cytometry analysis for CD36 and CD235a in combination with either CXCR4 (CD184), PSGL-1 (CD162), or VLA-4 (CD49d) of EBs obtained at day 10 of HEMA culture from AB and CB MNC (Prol 10) and at day 1 (Diff Day 1) and 4 (Diff Day 4) of maturation with EPO alone, as indicated. EBs were divided into 4 maturation classes defined by the levels of CD36 and CD235a expression identified by the gates R1 to R4 and corresponding to Class 1 to 4 (see Supplemental Figure 3 for further details).
Figure Legend Snippet: Adhesion receptor profiling during in vitro maturation of EBs expanded from AB (a) and CB (b). Three color flow cytometry analysis for CD36 and CD235a in combination with either CXCR4 (CD184), PSGL-1 (CD162), or VLA-4 (CD49d) of EBs obtained at day 10 of HEMA culture from AB and CB MNC (Prol 10) and at day 1 (Diff Day 1) and 4 (Diff Day 4) of maturation with EPO alone, as indicated. EBs were divided into 4 maturation classes defined by the levels of CD36 and CD235a expression identified by the gates R1 to R4 and corresponding to Class 1 to 4 (see Supplemental Figure 3 for further details).

Techniques Used: In Vitro, Flow Cytometry, Cytometry, Expressing

By flow cytometry of human CD36/CD235a expression, human CD235a pos cells were detectable in marrow and liver of splenectomized mice 4 days after transfusion. Flow cytometry analysis of bone marrow (left panels), spleen (middle panels) and liver (right panels) from splenectomized (top panels), non splenectomized (middle panels), and nontransfused controls (bottom panels) of representative mice are shown. The small numbers indicate the frequency of the cells detected within the respective quadrant. Values within circles correspond to the percentage of human cells calculated by subtracting the frequency detected in the negative controls in the corresponding bottom panels. The red circles indicate the frequency of human cells. Of note, the majority of the human CD235a positive cells were negative for CD36, an indication that the human EBs had matured in vivo . Similar results were obtained by analyzing the cells for eGFP expression. Data are representative of those obtained with 3 mice per experimental point.
Figure Legend Snippet: By flow cytometry of human CD36/CD235a expression, human CD235a pos cells were detectable in marrow and liver of splenectomized mice 4 days after transfusion. Flow cytometry analysis of bone marrow (left panels), spleen (middle panels) and liver (right panels) from splenectomized (top panels), non splenectomized (middle panels), and nontransfused controls (bottom panels) of representative mice are shown. The small numbers indicate the frequency of the cells detected within the respective quadrant. Values within circles correspond to the percentage of human cells calculated by subtracting the frequency detected in the negative controls in the corresponding bottom panels. The red circles indicate the frequency of human cells. Of note, the majority of the human CD235a positive cells were negative for CD36, an indication that the human EBs had matured in vivo . Similar results were obtained by analyzing the cells for eGFP expression. Data are representative of those obtained with 3 mice per experimental point.

Techniques Used: Flow Cytometry, Cytometry, Expressing, Mouse Assay, In Vivo

MNC from AB and CB generate great numbers of EBs under HEMA conditions. (a) Cell number (as Fold Increase, FI, with respect to day 0), (b) maturation profile (cytofluorimetric analysis on the basis of the expression of CD36 and CD235a), and (c) representative morphology (by May-Grunwald staining) of EBs generated in HEMA culture seeded with MNC from either CB or AB. The ability of CB and AB EBs obtained at day 10 to proceed along the maturation pathway after 4 days of culture in the presence of EPO only is also compared (b). The flow cytometric profile used to define EBs maturation is presented in Supplemental Figure 3. The flow charts are representative of those obtained in at least three independent experiments with MNC from different CB or AB donors. The numbers in the quadrants present the frequency of EBs in the gates R1 to R4. Frequencies obtained in multiple experiments are presented as mean (±SD) in Table 1 .
Figure Legend Snippet: MNC from AB and CB generate great numbers of EBs under HEMA conditions. (a) Cell number (as Fold Increase, FI, with respect to day 0), (b) maturation profile (cytofluorimetric analysis on the basis of the expression of CD36 and CD235a), and (c) representative morphology (by May-Grunwald staining) of EBs generated in HEMA culture seeded with MNC from either CB or AB. The ability of CB and AB EBs obtained at day 10 to proceed along the maturation pathway after 4 days of culture in the presence of EPO only is also compared (b). The flow cytometric profile used to define EBs maturation is presented in Supplemental Figure 3. The flow charts are representative of those obtained in at least three independent experiments with MNC from different CB or AB donors. The numbers in the quadrants present the frequency of EBs in the gates R1 to R4. Frequencies obtained in multiple experiments are presented as mean (±SD) in Table 1 .

Techniques Used: Expressing, Staining, Generated, Flow Cytometry

6) Product Images from "The BTK Inhibitor Ibrutinib (PCI-32765) Blocks Hairy Cell Leukaemia Survival, Proliferation and BCR Signalling: A New Therapeutic Approach"

Article Title: The BTK Inhibitor Ibrutinib (PCI-32765) Blocks Hairy Cell Leukaemia Survival, Proliferation and BCR Signalling: A New Therapeutic Approach

Journal: British journal of haematology

doi: 10.1111/bjh.12867

BCR-induced cell survival signalling in HCL cells is decreased after treatment with ibrutinib (A) Contour plots of a representative case, showing HCL cell viabilities after 48 h of incubation with anti Igs (anti IgA, anti IgG and anti IgM), following incubation for 1 h with ibrutinib (0.5 μM, 1 μM or 5 μM), as indicated below the plots. The gates in each plot highlight the viable cell population, defined as DiOC 6 positive and PI negative. (B) The left-hand graph represents the mean relative HCL cell viabilities after 24 and 48 h of incubation with anti Igs in the presence or absence of ibrutinib (indicated as control); the right-hand graph shows the XTT assay performed in parallel; results were normalized relative to the untrea1ted controls samples (100%). Displayed are the mean ± SEM, with ** as p
Figure Legend Snippet: BCR-induced cell survival signalling in HCL cells is decreased after treatment with ibrutinib (A) Contour plots of a representative case, showing HCL cell viabilities after 48 h of incubation with anti Igs (anti IgA, anti IgG and anti IgM), following incubation for 1 h with ibrutinib (0.5 μM, 1 μM or 5 μM), as indicated below the plots. The gates in each plot highlight the viable cell population, defined as DiOC 6 positive and PI negative. (B) The left-hand graph represents the mean relative HCL cell viabilities after 24 and 48 h of incubation with anti Igs in the presence or absence of ibrutinib (indicated as control); the right-hand graph shows the XTT assay performed in parallel; results were normalized relative to the untrea1ted controls samples (100%). Displayed are the mean ± SEM, with ** as p

Techniques Used: Incubation, XTT Assay

Ibrutinib down-regulates BCR signalling and inhibits the secretion of the chemokines CCL3 and CCL4 in HCL cells (A) Immunoblots from HCL primary cells (HCL13, HCL21 and HCL18) stimulated with anti Igs (anti IgA, anti IgG and anti IgM) for BCR triggering in the presence or absence of 0.1 μM, 0.5 μM or 1 μM ibrutinib, as indicated. P indicates immunoblots for the active phosphorylated form. GAPDH was used as a protein loading control. (B) The concentration of the chemokines CCL3 and CCL4 secreted in the supernatants of primary hairy cells was measured after BCR stimulation with anti Igs (anti IgA, anti IgG and anti IgM) in the presence or absence of ibrutinib as indicated, after 48 h of incubation. The bars represent the mean ± SEM (n=8) for CCL3 (left) and CCL4 (right).
Figure Legend Snippet: Ibrutinib down-regulates BCR signalling and inhibits the secretion of the chemokines CCL3 and CCL4 in HCL cells (A) Immunoblots from HCL primary cells (HCL13, HCL21 and HCL18) stimulated with anti Igs (anti IgA, anti IgG and anti IgM) for BCR triggering in the presence or absence of 0.1 μM, 0.5 μM or 1 μM ibrutinib, as indicated. P indicates immunoblots for the active phosphorylated form. GAPDH was used as a protein loading control. (B) The concentration of the chemokines CCL3 and CCL4 secreted in the supernatants of primary hairy cells was measured after BCR stimulation with anti Igs (anti IgA, anti IgG and anti IgM) in the presence or absence of ibrutinib as indicated, after 48 h of incubation. The bars represent the mean ± SEM (n=8) for CCL3 (left) and CCL4 (right).

Techniques Used: Western Blot, Concentration Assay, Incubation

Ibrutinib reduces phosphorylated BTK expression upon BCR stimulation in HCL cells (A) Immunoblots from 9 primary HCL cells, which were stimulated for 10 min with 10 μg/ml of anti Igs (anti IgA, anti IgG and anti IgM) in the presence or absence of 0.1 μM, 0.5 μM or 1 μM ibrutinib, as indicated. P indicates immunobloting for the active phosphorylated form of BTK (lower band). GAPDH was using as a loading control. (B) Occupancy test, assessed by the ability of BTK to bind to the fluorescence labelled probe (PCI-33380) in the presence of dose escalation of ibrutinib in the HCL cell lines, ESKOL and HC-1. Total BTK levels were determined by immunobloting. DOHH2 cell line lysates labelled with probe (100% and 10%) were used as a control.
Figure Legend Snippet: Ibrutinib reduces phosphorylated BTK expression upon BCR stimulation in HCL cells (A) Immunoblots from 9 primary HCL cells, which were stimulated for 10 min with 10 μg/ml of anti Igs (anti IgA, anti IgG and anti IgM) in the presence or absence of 0.1 μM, 0.5 μM or 1 μM ibrutinib, as indicated. P indicates immunobloting for the active phosphorylated form of BTK (lower band). GAPDH was using as a loading control. (B) Occupancy test, assessed by the ability of BTK to bind to the fluorescence labelled probe (PCI-33380) in the presence of dose escalation of ibrutinib in the HCL cell lines, ESKOL and HC-1. Total BTK levels were determined by immunobloting. DOHH2 cell line lysates labelled with probe (100% and 10%) were used as a control.

Techniques Used: Expressing, Western Blot, Fluorescence

Related Articles

Flow Cytometry:

Article Title: Human Fetal Liver Stromal Cells Expressing Erythropoietin Promote Hematopoietic Development from Human Embryonic Stem Cells
Article Snippet: .. The trypsinized individual cells were incubated with the FITC-conjugated and PE-conjugated monoclonal antibodies: antihuman CD29, antihuman CD105, antihuman CD44, antihuman CD90, antihuman CD34, and antihuman CD45 (BD Biosciences, San Jose, CA) at 4°C for 30 min. Then the cells were washed three times with PBS and analyzed by flow cytometry analysis using the FACSCalibur (Becton-Dickinson, Mountain View, CA). .. Hematopoietic colony assays were performed in 35-mm low-adhesion plastic dishes using MethoCult GF-H4434 semisolid medium (Stem Cell Technologies, Vancouver, Canada) consisting of 1% methylcellulose, 30% FBS, 1% bovine serum albumin (BSA), 50 ng/mL stem cell factor, 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF), 20 ng/mL granulocyte colony-stimulating factor (G-CSF), 20 ng/mL interleukin-3 (IL-3), 20 ng/mL interleukin-6 (IL-6), and 3 U/mL EPO.

Article Title: Insertional mutagenesis using the Sleeping Beauty transposon system identifies drivers of erythroleukemia in mice
Article Snippet: .. Flow cytometry analysis included a 4-color T-cell panel (CD8a-PE, CD4-APC, CD19-APC-Cy7and CD45r/B220-PerCP (BD Biosciences)) and a 6-color erythroid/myeloid panel (CD45-APC-Cy7, CD71-FITC, CD117-PE-Cy7, Ter119-APC, Ly-6G/Ly-6C (Gr1)-PerCP-Cy5.5, and B220-FITC (BD Biosciences) and CD3e Alexa Fluor 488 (BioLegend). .. Cells were also stained with DAPI to determine cell viability.

Article Title: Adoptive Transfer of Regulatory T Cells Protects against Coxsackievirus B3-Induced Cardiac Fibrosis
Article Snippet: .. Then, the cells were stained with PerCP-labeled anti-mouse CD4 (eBioscience) and FITC-conjugated anti-mouse CD25 (eBioscience) at 4 °C for 40 min. After washing, fixing and permeabilization, the cells were stained intracellularly with PE-conjugated anti-mouse Foxp3 (eBioscience) at 4 °C for 1 hour before by flow cytometry analysis on a FACS Calibur machine (BD Biosciences). .. Data were analyzed with CellQuest software (BD Biosciences).

Article Title: A Fusion Cytokine Coupling GMCSF to IL9 Induces Heterologous Receptor Clustering and STAT1 Hyperactivation through JAK2 Promiscuity
Article Snippet: .. Mast cell phenotype was confirmed by flow cytometry analysis with α-c-kit (BD Biosciences) and α-FcεRIα (Biolegend, San Diego, CA) antibodies. ..

Article Title: Revealing the alternative promoter usage of SAF/MAZ gene by bichromatic fluorescent reporter construct
Article Snippet: .. Although there were statistically meaningful differences amongst these four constructs for SAF-1 expression by flow cytometry analysis, the change of SAF-1 expression was not as obvious as for SAF-3 expression ( A) . ..

Article Title: Delphinidin, an active compound of red wine, inhibits endothelial cell apoptosis via nitric oxide pathway and regulation of calcium homeostasis
Article Snippet: .. PI (0.1 mg ml−1 ) was then added and samples were allowed to stand 15 min in the dark at room temperature before flow cytometry analysis using CELLQuest software (Becton Dickinson, San Jose, CA, U.S.A.). .. After incubation with actinomycin D, adherent cells were fixed for 10 min with PBS containing 3% paraformaldehyde on ice.

Article Title: Development of drug-inducible CRISPR-Cas9 systems for large-scale functional screening
Article Snippet: .. The cells were then treated with various concentrations of doxycycline (Sigma-Aldrich) or IPTG (Sigma-Aldrich) for at least 5 days before flow cytometry analysis using an LSRFortessa X20 instrument (BD Biosciences). .. Expression of sgRNA by RT-qPCR Total RNA was first extracted with RNeasy mini kit (QIAGEN) and treated with DNase I (QIAGEN) following the manufacturer’s instructions.

Cytometry:

Article Title: Human Fetal Liver Stromal Cells Expressing Erythropoietin Promote Hematopoietic Development from Human Embryonic Stem Cells
Article Snippet: .. The trypsinized individual cells were incubated with the FITC-conjugated and PE-conjugated monoclonal antibodies: antihuman CD29, antihuman CD105, antihuman CD44, antihuman CD90, antihuman CD34, and antihuman CD45 (BD Biosciences, San Jose, CA) at 4°C for 30 min. Then the cells were washed three times with PBS and analyzed by flow cytometry analysis using the FACSCalibur (Becton-Dickinson, Mountain View, CA). .. Hematopoietic colony assays were performed in 35-mm low-adhesion plastic dishes using MethoCult GF-H4434 semisolid medium (Stem Cell Technologies, Vancouver, Canada) consisting of 1% methylcellulose, 30% FBS, 1% bovine serum albumin (BSA), 50 ng/mL stem cell factor, 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF), 20 ng/mL granulocyte colony-stimulating factor (G-CSF), 20 ng/mL interleukin-3 (IL-3), 20 ng/mL interleukin-6 (IL-6), and 3 U/mL EPO.

Article Title: Insertional mutagenesis using the Sleeping Beauty transposon system identifies drivers of erythroleukemia in mice
Article Snippet: .. Flow cytometry analysis included a 4-color T-cell panel (CD8a-PE, CD4-APC, CD19-APC-Cy7and CD45r/B220-PerCP (BD Biosciences)) and a 6-color erythroid/myeloid panel (CD45-APC-Cy7, CD71-FITC, CD117-PE-Cy7, Ter119-APC, Ly-6G/Ly-6C (Gr1)-PerCP-Cy5.5, and B220-FITC (BD Biosciences) and CD3e Alexa Fluor 488 (BioLegend). .. Cells were also stained with DAPI to determine cell viability.

Article Title: Adoptive Transfer of Regulatory T Cells Protects against Coxsackievirus B3-Induced Cardiac Fibrosis
Article Snippet: .. Then, the cells were stained with PerCP-labeled anti-mouse CD4 (eBioscience) and FITC-conjugated anti-mouse CD25 (eBioscience) at 4 °C for 40 min. After washing, fixing and permeabilization, the cells were stained intracellularly with PE-conjugated anti-mouse Foxp3 (eBioscience) at 4 °C for 1 hour before by flow cytometry analysis on a FACS Calibur machine (BD Biosciences). .. Data were analyzed with CellQuest software (BD Biosciences).

Article Title: A Fusion Cytokine Coupling GMCSF to IL9 Induces Heterologous Receptor Clustering and STAT1 Hyperactivation through JAK2 Promiscuity
Article Snippet: .. Mast cell phenotype was confirmed by flow cytometry analysis with α-c-kit (BD Biosciences) and α-FcεRIα (Biolegend, San Diego, CA) antibodies. ..

Article Title: Revealing the alternative promoter usage of SAF/MAZ gene by bichromatic fluorescent reporter construct
Article Snippet: .. Although there were statistically meaningful differences amongst these four constructs for SAF-1 expression by flow cytometry analysis, the change of SAF-1 expression was not as obvious as for SAF-3 expression ( A) . ..

Article Title: Delphinidin, an active compound of red wine, inhibits endothelial cell apoptosis via nitric oxide pathway and regulation of calcium homeostasis
Article Snippet: .. PI (0.1 mg ml−1 ) was then added and samples were allowed to stand 15 min in the dark at room temperature before flow cytometry analysis using CELLQuest software (Becton Dickinson, San Jose, CA, U.S.A.). .. After incubation with actinomycin D, adherent cells were fixed for 10 min with PBS containing 3% paraformaldehyde on ice.

Article Title: Development of drug-inducible CRISPR-Cas9 systems for large-scale functional screening
Article Snippet: .. The cells were then treated with various concentrations of doxycycline (Sigma-Aldrich) or IPTG (Sigma-Aldrich) for at least 5 days before flow cytometry analysis using an LSRFortessa X20 instrument (BD Biosciences). .. Expression of sgRNA by RT-qPCR Total RNA was first extracted with RNeasy mini kit (QIAGEN) and treated with DNase I (QIAGEN) following the manufacturer’s instructions.

Construct:

Article Title: Revealing the alternative promoter usage of SAF/MAZ gene by bichromatic fluorescent reporter construct
Article Snippet: .. Although there were statistically meaningful differences amongst these four constructs for SAF-1 expression by flow cytometry analysis, the change of SAF-1 expression was not as obvious as for SAF-3 expression ( A) . ..

Incubation:

Article Title: Human Fetal Liver Stromal Cells Expressing Erythropoietin Promote Hematopoietic Development from Human Embryonic Stem Cells
Article Snippet: .. The trypsinized individual cells were incubated with the FITC-conjugated and PE-conjugated monoclonal antibodies: antihuman CD29, antihuman CD105, antihuman CD44, antihuman CD90, antihuman CD34, and antihuman CD45 (BD Biosciences, San Jose, CA) at 4°C for 30 min. Then the cells were washed three times with PBS and analyzed by flow cytometry analysis using the FACSCalibur (Becton-Dickinson, Mountain View, CA). .. Hematopoietic colony assays were performed in 35-mm low-adhesion plastic dishes using MethoCult GF-H4434 semisolid medium (Stem Cell Technologies, Vancouver, Canada) consisting of 1% methylcellulose, 30% FBS, 1% bovine serum albumin (BSA), 50 ng/mL stem cell factor, 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF), 20 ng/mL granulocyte colony-stimulating factor (G-CSF), 20 ng/mL interleukin-3 (IL-3), 20 ng/mL interleukin-6 (IL-6), and 3 U/mL EPO.

Expressing:

Article Title: Revealing the alternative promoter usage of SAF/MAZ gene by bichromatic fluorescent reporter construct
Article Snippet: .. Although there were statistically meaningful differences amongst these four constructs for SAF-1 expression by flow cytometry analysis, the change of SAF-1 expression was not as obvious as for SAF-3 expression ( A) . ..

Staining:

Article Title: Adoptive Transfer of Regulatory T Cells Protects against Coxsackievirus B3-Induced Cardiac Fibrosis
Article Snippet: .. Then, the cells were stained with PerCP-labeled anti-mouse CD4 (eBioscience) and FITC-conjugated anti-mouse CD25 (eBioscience) at 4 °C for 40 min. After washing, fixing and permeabilization, the cells were stained intracellularly with PE-conjugated anti-mouse Foxp3 (eBioscience) at 4 °C for 1 hour before by flow cytometry analysis on a FACS Calibur machine (BD Biosciences). .. Data were analyzed with CellQuest software (BD Biosciences).

FACS:

Article Title: Adoptive Transfer of Regulatory T Cells Protects against Coxsackievirus B3-Induced Cardiac Fibrosis
Article Snippet: .. Then, the cells were stained with PerCP-labeled anti-mouse CD4 (eBioscience) and FITC-conjugated anti-mouse CD25 (eBioscience) at 4 °C for 40 min. After washing, fixing and permeabilization, the cells were stained intracellularly with PE-conjugated anti-mouse Foxp3 (eBioscience) at 4 °C for 1 hour before by flow cytometry analysis on a FACS Calibur machine (BD Biosciences). .. Data were analyzed with CellQuest software (BD Biosciences).

Software:

Article Title: Delphinidin, an active compound of red wine, inhibits endothelial cell apoptosis via nitric oxide pathway and regulation of calcium homeostasis
Article Snippet: .. PI (0.1 mg ml−1 ) was then added and samples were allowed to stand 15 min in the dark at room temperature before flow cytometry analysis using CELLQuest software (Becton Dickinson, San Jose, CA, U.S.A.). .. After incubation with actinomycin D, adherent cells were fixed for 10 min with PBS containing 3% paraformaldehyde on ice.

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