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Miltenyi Biotec anti cd19 apc
iCASP9 suicide system functional assay with the IL-1RAP low,inter or high CAR+ leukemic AML cell lines or patient-derived xenograft (PDX) AML blasts. Untransduced (UT, open symbols, clear bars) or CAR-transduced (CAR+, closed symbols, grey bars) leukemic AML cells were cultured in medium alone or medium containing 20 nM Rimiducid. After 24 h of Rimiducid exposure, cells were collected and transferred to Trucount tubes and stained with <t>anti-CD19,</t> anti-Annexin-V, and 7-aminoactinomycin D (7-AAD). a Gating strategy for cytometry for discrimination of viable from dead cells. The quantification was performed after acquiring 5000 fluorescent beads. The mortality rate was normalized to control cells (untreated cells) and calculated as follows: % Dead cells = [1 − (absolute number of viable cells in AP1903-treated cells/absolute number of viable cells in untreated cells)] × 100. b Mortality results are shown as mean ± SD from triplicate (cell lines) or quadruplicate (PDX AML blasts) samples for each condition. **** p
Anti Cd19 Apc, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Overcoming target epitope masking resistance that can occur on low-antigen-expresser AML blasts after IL-1RAP chimeric antigen receptor T cell therapy using the inducible caspase 9 suicide gene safety switch"

Article Title: Overcoming target epitope masking resistance that can occur on low-antigen-expresser AML blasts after IL-1RAP chimeric antigen receptor T cell therapy using the inducible caspase 9 suicide gene safety switch

Journal: Cancer Gene Therapy

doi: 10.1038/s41417-020-00284-3

iCASP9 suicide system functional assay with the IL-1RAP low,inter or high CAR+ leukemic AML cell lines or patient-derived xenograft (PDX) AML blasts. Untransduced (UT, open symbols, clear bars) or CAR-transduced (CAR+, closed symbols, grey bars) leukemic AML cells were cultured in medium alone or medium containing 20 nM Rimiducid. After 24 h of Rimiducid exposure, cells were collected and transferred to Trucount tubes and stained with anti-CD19, anti-Annexin-V, and 7-aminoactinomycin D (7-AAD). a Gating strategy for cytometry for discrimination of viable from dead cells. The quantification was performed after acquiring 5000 fluorescent beads. The mortality rate was normalized to control cells (untreated cells) and calculated as follows: % Dead cells = [1 − (absolute number of viable cells in AP1903-treated cells/absolute number of viable cells in untreated cells)] × 100. b Mortality results are shown as mean ± SD from triplicate (cell lines) or quadruplicate (PDX AML blasts) samples for each condition. **** p
Figure Legend Snippet: iCASP9 suicide system functional assay with the IL-1RAP low,inter or high CAR+ leukemic AML cell lines or patient-derived xenograft (PDX) AML blasts. Untransduced (UT, open symbols, clear bars) or CAR-transduced (CAR+, closed symbols, grey bars) leukemic AML cells were cultured in medium alone or medium containing 20 nM Rimiducid. After 24 h of Rimiducid exposure, cells were collected and transferred to Trucount tubes and stained with anti-CD19, anti-Annexin-V, and 7-aminoactinomycin D (7-AAD). a Gating strategy for cytometry for discrimination of viable from dead cells. The quantification was performed after acquiring 5000 fluorescent beads. The mortality rate was normalized to control cells (untreated cells) and calculated as follows: % Dead cells = [1 − (absolute number of viable cells in AP1903-treated cells/absolute number of viable cells in untreated cells)] × 100. b Mortality results are shown as mean ± SD from triplicate (cell lines) or quadruplicate (PDX AML blasts) samples for each condition. **** p

Techniques Used: Functional Assay, Derivative Assay, Cell Culture, Staining, Cytometry

Generation of CAR-transduced leukemic cell lines. a Determination of the absolute number of IL-1RAP antigenic sites on the surface of seven different AML cell lines by flow cytometry using calibration beads, using anti-IL-1RAP antibody and a secondary anti-mouse antibody coupled to FITC. Three groups were defined by IL-1RAP expression level: IL-1RAP low (HEL, HL-60, and MA9RAS), IL-1RAP inter (EOL-1, Molm-13, and THP-1), and IL-1RAP high (Mono-Mac-6 and KU812). K562 and KU812 cell lines were used as negative and positive controls, respectively. b Production of CAR+ leukemic AML cell lines. Cell lines belonging to the three IL-1RAP expression-level groups were transduced with mock or CAR vector. Transduction efficiency, provided as a percentage, was measured by flow cytometry using anti-CD19-APC, representing cell surface CD19 expression on transduced cells. Untransduced cells were used as a negative control. c Upper: normalization by gating on UT, Mock, or CAR transduced cell line subpopulations based on similar CD19 cell surface expression (grey area). Left lower: CAR expression at the cell surface of CAR-transduced leukemic cells. Representative experiment: cells were incubated with biotinylated recombinant IL-1RAP protein and stained using streptavidin-PE by flow cytometry. RFI of IL-1RAP CAR staining relative to UT cells is provided. Dark gray represents the untransduced cells. Light gray, blue, or red histograms correspond, respectively, to matched isotype staining or Biot-IL-1RAP staining of Mock- or CAR-transduced cell lines. Right lower: relative cell surface expression of IL‐1RAP on AML cell lines HL‐60, Molm‐13, and Mono‐Mac‐6 before (untransduced = UT) or after transduction with mock or CAR vectors determined by staining cells with anti‐IL‐1RAP‐FITC and flow cytometry. An isotype-matched IgG‐FITC was used as a negative control. MFI (mean of fluorescence intensity) is provided. d Relative expression of IL-1RAP CAR cell surface expression, expressed as relative fluorescence intensity (RFI) on the three different cell lines before (untransduced, blue histograms) or after transduction with mock (green histograms) or CAR vector (red histograms) determined by staining cells with anti-IL-1RAP-FITC and flow cytometry. e Relative expression of IL-1RAP cell surface expression, expressed as relative fluorescence intensity (RFI) on the cell lines HL-60 (IL-1RAP low), Molm-13 (IL-1RAP int), and Mono-Mac-6 (IL-1RAP high) cells before (untransduced, blue histograms) or after transduction with mock (green histograms) or CAR vector (red histograms) determined by staining cells with anti-IL-1RAP-FITC and flow cytometry. Analysis was performed by gating on similar CD19 expresser AML subpopulations. An isotype-matched IgG-FITC was used as a negative control. Graph represents four independent experiments. ** p
Figure Legend Snippet: Generation of CAR-transduced leukemic cell lines. a Determination of the absolute number of IL-1RAP antigenic sites on the surface of seven different AML cell lines by flow cytometry using calibration beads, using anti-IL-1RAP antibody and a secondary anti-mouse antibody coupled to FITC. Three groups were defined by IL-1RAP expression level: IL-1RAP low (HEL, HL-60, and MA9RAS), IL-1RAP inter (EOL-1, Molm-13, and THP-1), and IL-1RAP high (Mono-Mac-6 and KU812). K562 and KU812 cell lines were used as negative and positive controls, respectively. b Production of CAR+ leukemic AML cell lines. Cell lines belonging to the three IL-1RAP expression-level groups were transduced with mock or CAR vector. Transduction efficiency, provided as a percentage, was measured by flow cytometry using anti-CD19-APC, representing cell surface CD19 expression on transduced cells. Untransduced cells were used as a negative control. c Upper: normalization by gating on UT, Mock, or CAR transduced cell line subpopulations based on similar CD19 cell surface expression (grey area). Left lower: CAR expression at the cell surface of CAR-transduced leukemic cells. Representative experiment: cells were incubated with biotinylated recombinant IL-1RAP protein and stained using streptavidin-PE by flow cytometry. RFI of IL-1RAP CAR staining relative to UT cells is provided. Dark gray represents the untransduced cells. Light gray, blue, or red histograms correspond, respectively, to matched isotype staining or Biot-IL-1RAP staining of Mock- or CAR-transduced cell lines. Right lower: relative cell surface expression of IL‐1RAP on AML cell lines HL‐60, Molm‐13, and Mono‐Mac‐6 before (untransduced = UT) or after transduction with mock or CAR vectors determined by staining cells with anti‐IL‐1RAP‐FITC and flow cytometry. An isotype-matched IgG‐FITC was used as a negative control. MFI (mean of fluorescence intensity) is provided. d Relative expression of IL-1RAP CAR cell surface expression, expressed as relative fluorescence intensity (RFI) on the three different cell lines before (untransduced, blue histograms) or after transduction with mock (green histograms) or CAR vector (red histograms) determined by staining cells with anti-IL-1RAP-FITC and flow cytometry. e Relative expression of IL-1RAP cell surface expression, expressed as relative fluorescence intensity (RFI) on the cell lines HL-60 (IL-1RAP low), Molm-13 (IL-1RAP int), and Mono-Mac-6 (IL-1RAP high) cells before (untransduced, blue histograms) or after transduction with mock (green histograms) or CAR vector (red histograms) determined by staining cells with anti-IL-1RAP-FITC and flow cytometry. Analysis was performed by gating on similar CD19 expresser AML subpopulations. An isotype-matched IgG-FITC was used as a negative control. Graph represents four independent experiments. ** p

Techniques Used: Flow Cytometry, Expressing, Transduction, Plasmid Preparation, Negative Control, Incubation, Recombinant, Staining, Fluorescence

In vitro cytotoxicity of IL-1RAP CAR T cells against CAR+ leukemic cells. Untransduced, mock (circle, dotted line), or CAR T cells (square, solid line) as effector cells were labeled with eFluor and co-cultured with each cell line (HL-60, Molm-13, and Mono-Mac-6 cells) untransduced (UT tumor, white symbols) or transduced with mock (mock tumor, gray symbols) or CAR (CAR tumor, black/red symbols) vectors, at various effector:target ratios. Effector cytotoxicity is reported as the percentage of remaining living cells, gated in cytometry as eFluor−/7-AAD−, normalized to UT effectors. Results are presented as mean ± SD of three independent experiments. Analysis was performed by gating on similar CD19 expresser AML subpopulations. Comparison of IL-1RAP CAR T cells cytotoxicity against UT or Mock versus IL-1RAP CAR+ transduced low IL-1RAP HL-60 AML tumor cell lines: ** p
Figure Legend Snippet: In vitro cytotoxicity of IL-1RAP CAR T cells against CAR+ leukemic cells. Untransduced, mock (circle, dotted line), or CAR T cells (square, solid line) as effector cells were labeled with eFluor and co-cultured with each cell line (HL-60, Molm-13, and Mono-Mac-6 cells) untransduced (UT tumor, white symbols) or transduced with mock (mock tumor, gray symbols) or CAR (CAR tumor, black/red symbols) vectors, at various effector:target ratios. Effector cytotoxicity is reported as the percentage of remaining living cells, gated in cytometry as eFluor−/7-AAD−, normalized to UT effectors. Results are presented as mean ± SD of three independent experiments. Analysis was performed by gating on similar CD19 expresser AML subpopulations. Comparison of IL-1RAP CAR T cells cytotoxicity against UT or Mock versus IL-1RAP CAR+ transduced low IL-1RAP HL-60 AML tumor cell lines: ** p

Techniques Used: In Vitro, Labeling, Cell Culture, Transduction, Cytometry

2) Product Images from "Memory persistence and differentiation into antibody-secreting cells accompanied by positive selection in longitudinal BCR repertoires"

Article Title: Memory persistence and differentiation into antibody-secreting cells accompanied by positive selection in longitudinal BCR repertoires

Journal: bioRxiv

doi: 10.1101/2021.12.30.474135

A : FACS gating strategy and the frequencies of studied cell subsets for representative peripheral blood sample (donor IZ time point T3): Memory B-cells (Bmem: CD19 + CD20 + CD27 + ), plasmablasts (PBL: CD19 low/+ CD20 - CD27 high CD138 - ) and plasma cells (PL: CD19 low/+ CD20 - CD27 high CD138 + ); B : Isotype frequencies for individual samples by unique clonotypes. Whiskers illustrate minimal and maximal isotype frequencies for the group. Black and grey lines at the bottom of the plot indicate groups of bars corresponding to a particular donor.
Figure Legend Snippet: A : FACS gating strategy and the frequencies of studied cell subsets for representative peripheral blood sample (donor IZ time point T3): Memory B-cells (Bmem: CD19 + CD20 + CD27 + ), plasmablasts (PBL: CD19 low/+ CD20 - CD27 high CD138 - ) and plasma cells (PL: CD19 low/+ CD20 - CD27 high CD138 + ); B : Isotype frequencies for individual samples by unique clonotypes. Whiskers illustrate minimal and maximal isotype frequencies for the group. Black and grey lines at the bottom of the plot indicate groups of bars corresponding to a particular donor.

Techniques Used: FACS

General characteristics of IGH repertoires in differentiated B-cell lineage subsets. A : Study design. Peripheral blood of 6 donors was sampled at three time points: T1 - initial time point, T2 −1 month and T3 - 12 months later from the start of the study. At each time point we isolated PBMCs and sorted memory B cells (Bmem: CD19 + CD20 + CD27 + ), plasmablasts (PBL: CD19 low/+ CD20 - CD27 high CD138-) and plasma cells (PL: CD19 low/+ CD20 - CD27 high CD138 + ) in two replicates using FACS. For each cell sample we obtained clonal repertoires of full-length IGH by sequencing of respective cDNA libraries; B : Proportion of isotypes in studied cell subsets averaged across all obtained repertoires. Left panel - frequency of unique IGH clonotypes with each particular isotype. Right panel - frequency of each isotype by IGH cDNA molecules detected in a sample; C : Distribution of the number of somatic hypermutations identified per 100 bp length of IGHV-segment for clonotypes with each particular isotype; D : Distribution of CDR3 length of clonotypes in studied cell subsets by isotype; E : Distributions of average IGHV gene frequencies by number of clonotypes of naive B-cell ( Gidoni et al. 2019 ), memory B-cell, plasmablast and plasma cell repertoires are shown at the top. Colored squares on heatmap indicate significantly different (FDR
Figure Legend Snippet: General characteristics of IGH repertoires in differentiated B-cell lineage subsets. A : Study design. Peripheral blood of 6 donors was sampled at three time points: T1 - initial time point, T2 −1 month and T3 - 12 months later from the start of the study. At each time point we isolated PBMCs and sorted memory B cells (Bmem: CD19 + CD20 + CD27 + ), plasmablasts (PBL: CD19 low/+ CD20 - CD27 high CD138-) and plasma cells (PL: CD19 low/+ CD20 - CD27 high CD138 + ) in two replicates using FACS. For each cell sample we obtained clonal repertoires of full-length IGH by sequencing of respective cDNA libraries; B : Proportion of isotypes in studied cell subsets averaged across all obtained repertoires. Left panel - frequency of unique IGH clonotypes with each particular isotype. Right panel - frequency of each isotype by IGH cDNA molecules detected in a sample; C : Distribution of the number of somatic hypermutations identified per 100 bp length of IGHV-segment for clonotypes with each particular isotype; D : Distribution of CDR3 length of clonotypes in studied cell subsets by isotype; E : Distributions of average IGHV gene frequencies by number of clonotypes of naive B-cell ( Gidoni et al. 2019 ), memory B-cell, plasmablast and plasma cell repertoires are shown at the top. Colored squares on heatmap indicate significantly different (FDR

Techniques Used: Isolation, FACS, Sequencing

3) Product Images from "Next-Generation Sequencing Revealed a Distinct Immunoglobulin Repertoire with Specific Mutation Hotspots in Acute Myeloid Leukemia"

Article Title: Next-Generation Sequencing Revealed a Distinct Immunoglobulin Repertoire with Specific Mutation Hotspots in Acute Myeloid Leukemia

Journal: Biology

doi: 10.3390/biology11020161

Expression of IGHG and IGKC in AML blasts. ( A ) Schematic map for flow cytometry sorting strategy of AML samples. Blasts: CD45 dim SSC low , lym: CD45 high SSC low lymphocytes. CD19 was further used to exclude B cell contamination from AML blasts. ( B ) Schematic map of Ig structure and primers complementary to IgG and Igκ constant region used for qPCR analysis. IGHV: variable region of Ig heavy chain, VL: variable region of Ig light chain, CH: constant region of Ig heavy chain, CL: constant region of Ig light chain, FR: framework regions, CDR: complementary determining regions. ( C ) Spearman correlation analysis shows a strong correlation between levels of IGHG and IGKC expression. ( D ) Kaplan–Meier analysis shows higher levels of IGHG expression correlate with shorter disease-free survival in AML patients.
Figure Legend Snippet: Expression of IGHG and IGKC in AML blasts. ( A ) Schematic map for flow cytometry sorting strategy of AML samples. Blasts: CD45 dim SSC low , lym: CD45 high SSC low lymphocytes. CD19 was further used to exclude B cell contamination from AML blasts. ( B ) Schematic map of Ig structure and primers complementary to IgG and Igκ constant region used for qPCR analysis. IGHV: variable region of Ig heavy chain, VL: variable region of Ig light chain, CH: constant region of Ig heavy chain, CL: constant region of Ig light chain, FR: framework regions, CDR: complementary determining regions. ( C ) Spearman correlation analysis shows a strong correlation between levels of IGHG and IGKC expression. ( D ) Kaplan–Meier analysis shows higher levels of IGHG expression correlate with shorter disease-free survival in AML patients.

Techniques Used: Expressing, Flow Cytometry, Real-time Polymerase Chain Reaction

4) Product Images from "Inhibition of B cell–dependent lymphoid follicle formation prevents lymphocytic bronchiolitis after lung transplantation"

Article Title: Inhibition of B cell–dependent lymphoid follicle formation prevents lymphocytic bronchiolitis after lung transplantation

Journal: JCI Insight

doi: 10.1172/jci.insight.123971

HLA-A2–knockin lung allografts contain follicles and activated B cells. Left lungs from C57BL/6J (B6) and HLA-A2–knockin (HLA) mice on a B6 background (HLA) were orthotopically transplanted into B6 recipient mice and analyzed 2 months after LTx (B6→B6, n = 4, HLA→B6, n = 4). ( A ), and counterstained with the nuclear marker DAPI. CD3 + and CD19 + cells infiltrating the grafts, analyzed by flow cytometry (FACS), 2 months after LTx. Representative FACS plots and quantification of CD3 + and CD19 + cells, as percentage of live cells, are shown. Data are expressed as mean ± SEM and were analyzed with a Mann-Whitney test; * P
Figure Legend Snippet: HLA-A2–knockin lung allografts contain follicles and activated B cells. Left lungs from C57BL/6J (B6) and HLA-A2–knockin (HLA) mice on a B6 background (HLA) were orthotopically transplanted into B6 recipient mice and analyzed 2 months after LTx (B6→B6, n = 4, HLA→B6, n = 4). ( A ), and counterstained with the nuclear marker DAPI. CD3 + and CD19 + cells infiltrating the grafts, analyzed by flow cytometry (FACS), 2 months after LTx. Representative FACS plots and quantification of CD3 + and CD19 + cells, as percentage of live cells, are shown. Data are expressed as mean ± SEM and were analyzed with a Mann-Whitney test; * P

Techniques Used: Knock-In, Mouse Assay, Marker, Flow Cytometry, FACS, MANN-WHITNEY

HLA-A2–knockin lung allografts contain germinal centers and plasma cells. Left lungs from C57BL/6J (B6) and HLA-A2–knockin (HLA) mice on a B6 background (HLA) orthotopically transplanted into B6 recipient mice. The mice were analyzed 2 months after LTx (B6→B6, n = 4, HLA→B6, n = 4). ( A ) and counterstained with the nuclear marker DAPI. Bottom: Flow cytometry analysis of the GL7 + B cells infiltrating the lung grafts. Left: Representative FACS plots of GL7 fluorescence plotted against cell size (FSC-A) and gated on CD19 + cells. Right: Quantification of GL7 + cells, expressed as a percentage of CD19 + cells. Data are expressed as mean ± SEM and analyzed with a Mann-Whitney test. * P
Figure Legend Snippet: HLA-A2–knockin lung allografts contain germinal centers and plasma cells. Left lungs from C57BL/6J (B6) and HLA-A2–knockin (HLA) mice on a B6 background (HLA) orthotopically transplanted into B6 recipient mice. The mice were analyzed 2 months after LTx (B6→B6, n = 4, HLA→B6, n = 4). ( A ) and counterstained with the nuclear marker DAPI. Bottom: Flow cytometry analysis of the GL7 + B cells infiltrating the lung grafts. Left: Representative FACS plots of GL7 fluorescence plotted against cell size (FSC-A) and gated on CD19 + cells. Right: Quantification of GL7 + cells, expressed as a percentage of CD19 + cells. Data are expressed as mean ± SEM and analyzed with a Mann-Whitney test. * P

Techniques Used: Knock-In, Mouse Assay, Marker, Flow Cytometry, FACS, Fluorescence, MANN-WHITNEY

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    Miltenyi Biotec apc conjugated anti cd19 antibody
    ET-1 stimulation induces a pro-angiogenic profile in CLL cells (A) Box plot represents the MFIR of HIF-1α in 7 CLL samples either stimulated or not stimulated with ET-1 for 30 minutes. On the right, histograms show the fluorescence intensity of <t>CD19+</t> CLL cells after treatment with ET-1 stained with anti-HIF-1α antibody. (B) CLL cells were allowed to adhere on the coverslip and then stimulated for 30 minutes with ET-1. HIF-1α induction was analyzed by immunofluorescence microscopy. Three representative CLL samples are shown. On the right, bar diagram represents quantification of cell staining, as mean value obtained from 5 different fields at 400X magnification normalized on control (100%, untreated sample). (C) CD19+ CLL cells were stimulated or not with ET-1 either in presence or absence of a pre-treatment with macitentan. Bar diagram depicts VEGF transcriptional levels (n=5, * P
    Apc Conjugated Anti Cd19 Antibody, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/apc conjugated anti cd19 antibody/product/Miltenyi Biotec
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    Miltenyi Biotec cd19 antibody anti human reafinity
    Study design, biospecimens, B-cell purification, molecular profiling, experiments and data analysis workflow. A) Lymph node biopsies and peripheral blood were collected from BCL patients (18 FL, 11 DLBCL, 23 CLL) and tonsils [36] from healthy donors. We purified <t>CD19</t> + malignant B cells from lymph node biopsies (FL, CLL, DLBCL) or peripheral blood (CLL), and isolated CD19 + normal B-cells from tonsils [20] . From additional tonsils we sorted germinal center (GC) centrocytes (CD19 + CD10 + CD44loCXCR4 − , 5), GC centroblasts (CD19 + CD10 + CD44loCXCR4 + , 5), naive (CD19 + CD5 − CD27 − , 3), and memory (CD19 + CD5 − CD27 + , 3) control B-cell subsets. From these samples, we performed chromatin immunoprecipitation sequencing (ChIPseq, H3K27ac – 51, H3ac – 47, H3K4me1 – 35)), open chromatin profiling (FAIREseq - 45), RNA sequencing (RNAseq - 28) and microarrays [80] , and whole genome copy number studies (SNP microarray - 42), totaling 328 high resolution molecular profiling studies. Our Integrative Analysis pipeline compared BCL subtypes to healthy control B-cells and identified copy number alterations, differentially bound enhancers, differentially expressed genes, and enhancer-gene associations. These studies also identified > 1300 super-enhancers, many of which correlate with significantly altered expression of neighboring genes in BCL compared to control B-cells. We validated one of these, a novel super-enhancer with high levels of epigenetic activity and expression of two nearby genes, FCMR and PIGR , across BCL subtypes, using luciferase reporter assays and demonstrate high levels of FCMR and PIGR protein in primary BCL cells using flow cytometry and immunofluorescence (IF) staining. B) Heatmap shows assays performed for each sample: healthy control (HC) CD19+ B cells; NAIVE B cells; Memory B cells (MEM); Centroblast (CB); Centrocyte (CC); CLL; DLBCL; FL.
    Cd19 Antibody Anti Human Reafinity, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cd19 antibody anti human reafinity/product/Miltenyi Biotec
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    Price from $9.99 to $1999.99
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    92
    Miltenyi Biotec cd19 apc
    <t>CD19/CD5</t> positive B-CLL cells isolated from CLL1C (month 8, panel A ) and CLL5C (month 24, panel B ) patients were stained with FITC-conjugated peptide p1 or unstained. p1-positive cells corresponding to the VH 1-69 clones were 70% of total B-CLL population in CLL1C patient and 32% in CLL5C patient.
    Cd19 Apc, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ET-1 stimulation induces a pro-angiogenic profile in CLL cells (A) Box plot represents the MFIR of HIF-1α in 7 CLL samples either stimulated or not stimulated with ET-1 for 30 minutes. On the right, histograms show the fluorescence intensity of CD19+ CLL cells after treatment with ET-1 stained with anti-HIF-1α antibody. (B) CLL cells were allowed to adhere on the coverslip and then stimulated for 30 minutes with ET-1. HIF-1α induction was analyzed by immunofluorescence microscopy. Three representative CLL samples are shown. On the right, bar diagram represents quantification of cell staining, as mean value obtained from 5 different fields at 400X magnification normalized on control (100%, untreated sample). (C) CD19+ CLL cells were stimulated or not with ET-1 either in presence or absence of a pre-treatment with macitentan. Bar diagram depicts VEGF transcriptional levels (n=5, * P

    Journal: Oncotarget

    Article Title: Macitentan, a double antagonist of endothelin receptors, efficiently impairs migration and microenvironmental survival signals in chronic lymphocytic leukemia

    doi: 10.18632/oncotarget.21341

    Figure Lengend Snippet: ET-1 stimulation induces a pro-angiogenic profile in CLL cells (A) Box plot represents the MFIR of HIF-1α in 7 CLL samples either stimulated or not stimulated with ET-1 for 30 minutes. On the right, histograms show the fluorescence intensity of CD19+ CLL cells after treatment with ET-1 stained with anti-HIF-1α antibody. (B) CLL cells were allowed to adhere on the coverslip and then stimulated for 30 minutes with ET-1. HIF-1α induction was analyzed by immunofluorescence microscopy. Three representative CLL samples are shown. On the right, bar diagram represents quantification of cell staining, as mean value obtained from 5 different fields at 400X magnification normalized on control (100%, untreated sample). (C) CD19+ CLL cells were stimulated or not with ET-1 either in presence or absence of a pre-treatment with macitentan. Bar diagram depicts VEGF transcriptional levels (n=5, * P

    Article Snippet: Then, CLL firmly adherent to HUVEC, 3T3 and HS-5 layer were counted by staining with APC-conjugated anti-CD19 antibody (Miltenyi Biotec) as previously described [ ].

    Techniques: Fluorescence, Staining, Immunofluorescence, Microscopy

    Macitentan affects CLL proliferation mediated by contact with stromal cells (A) CFSE-labeled CLL cells were cultured for 4 days alone in complete medium (control) or on 3T3 murine stromal cell layers. Where indicated, CLL cells were incubated for 1 hour with macitentan before co-culture. The proliferative measure was inspected for 4 days, gating the CD19+ live CLL cells. The histograms represent cumulative data at 96 hours of 3 independent experiments by using 3 CLL patients. Data are shown as mean values±SEM of the percentage of dividing CLL cells. The percentage of dividing CLL cells cultured alone was irrelevant. (B) CLL cells were either pre-treated or not with macitentan for 1 hour and then cultured for 24 hours. Panel B shows two representative western blots blotted for pGSK3β and β-catenin. Bar diagrams depict densitometric quantification of bands relative to pGSK3β/total GSK3β and β-catenin either in presence or absence of macitentan, normalized on β-actin. Data are presented as mean ± SEM of 4 different CLL samples ( * P

    Journal: Oncotarget

    Article Title: Macitentan, a double antagonist of endothelin receptors, efficiently impairs migration and microenvironmental survival signals in chronic lymphocytic leukemia

    doi: 10.18632/oncotarget.21341

    Figure Lengend Snippet: Macitentan affects CLL proliferation mediated by contact with stromal cells (A) CFSE-labeled CLL cells were cultured for 4 days alone in complete medium (control) or on 3T3 murine stromal cell layers. Where indicated, CLL cells were incubated for 1 hour with macitentan before co-culture. The proliferative measure was inspected for 4 days, gating the CD19+ live CLL cells. The histograms represent cumulative data at 96 hours of 3 independent experiments by using 3 CLL patients. Data are shown as mean values±SEM of the percentage of dividing CLL cells. The percentage of dividing CLL cells cultured alone was irrelevant. (B) CLL cells were either pre-treated or not with macitentan for 1 hour and then cultured for 24 hours. Panel B shows two representative western blots blotted for pGSK3β and β-catenin. Bar diagrams depict densitometric quantification of bands relative to pGSK3β/total GSK3β and β-catenin either in presence or absence of macitentan, normalized on β-actin. Data are presented as mean ± SEM of 4 different CLL samples ( * P

    Article Snippet: Then, CLL firmly adherent to HUVEC, 3T3 and HS-5 layer were counted by staining with APC-conjugated anti-CD19 antibody (Miltenyi Biotec) as previously described [ ].

    Techniques: Labeling, Cell Culture, Incubation, Co-Culture Assay, Western Blot

    Study design, biospecimens, B-cell purification, molecular profiling, experiments and data analysis workflow. A) Lymph node biopsies and peripheral blood were collected from BCL patients (18 FL, 11 DLBCL, 23 CLL) and tonsils [36] from healthy donors. We purified CD19 + malignant B cells from lymph node biopsies (FL, CLL, DLBCL) or peripheral blood (CLL), and isolated CD19 + normal B-cells from tonsils [20] . From additional tonsils we sorted germinal center (GC) centrocytes (CD19 + CD10 + CD44loCXCR4 − , 5), GC centroblasts (CD19 + CD10 + CD44loCXCR4 + , 5), naive (CD19 + CD5 − CD27 − , 3), and memory (CD19 + CD5 − CD27 + , 3) control B-cell subsets. From these samples, we performed chromatin immunoprecipitation sequencing (ChIPseq, H3K27ac – 51, H3ac – 47, H3K4me1 – 35)), open chromatin profiling (FAIREseq - 45), RNA sequencing (RNAseq - 28) and microarrays [80] , and whole genome copy number studies (SNP microarray - 42), totaling 328 high resolution molecular profiling studies. Our Integrative Analysis pipeline compared BCL subtypes to healthy control B-cells and identified copy number alterations, differentially bound enhancers, differentially expressed genes, and enhancer-gene associations. These studies also identified > 1300 super-enhancers, many of which correlate with significantly altered expression of neighboring genes in BCL compared to control B-cells. We validated one of these, a novel super-enhancer with high levels of epigenetic activity and expression of two nearby genes, FCMR and PIGR , across BCL subtypes, using luciferase reporter assays and demonstrate high levels of FCMR and PIGR protein in primary BCL cells using flow cytometry and immunofluorescence (IF) staining. B) Heatmap shows assays performed for each sample: healthy control (HC) CD19+ B cells; NAIVE B cells; Memory B cells (MEM); Centroblast (CB); Centrocyte (CC); CLL; DLBCL; FL.

    Journal: EBioMedicine

    Article Title: Loss of synergistic transcriptional feedback loops drives diverse B-cell cancers

    doi: 10.1016/j.ebiom.2021.103559

    Figure Lengend Snippet: Study design, biospecimens, B-cell purification, molecular profiling, experiments and data analysis workflow. A) Lymph node biopsies and peripheral blood were collected from BCL patients (18 FL, 11 DLBCL, 23 CLL) and tonsils [36] from healthy donors. We purified CD19 + malignant B cells from lymph node biopsies (FL, CLL, DLBCL) or peripheral blood (CLL), and isolated CD19 + normal B-cells from tonsils [20] . From additional tonsils we sorted germinal center (GC) centrocytes (CD19 + CD10 + CD44loCXCR4 − , 5), GC centroblasts (CD19 + CD10 + CD44loCXCR4 + , 5), naive (CD19 + CD5 − CD27 − , 3), and memory (CD19 + CD5 − CD27 + , 3) control B-cell subsets. From these samples, we performed chromatin immunoprecipitation sequencing (ChIPseq, H3K27ac – 51, H3ac – 47, H3K4me1 – 35)), open chromatin profiling (FAIREseq - 45), RNA sequencing (RNAseq - 28) and microarrays [80] , and whole genome copy number studies (SNP microarray - 42), totaling 328 high resolution molecular profiling studies. Our Integrative Analysis pipeline compared BCL subtypes to healthy control B-cells and identified copy number alterations, differentially bound enhancers, differentially expressed genes, and enhancer-gene associations. These studies also identified > 1300 super-enhancers, many of which correlate with significantly altered expression of neighboring genes in BCL compared to control B-cells. We validated one of these, a novel super-enhancer with high levels of epigenetic activity and expression of two nearby genes, FCMR and PIGR , across BCL subtypes, using luciferase reporter assays and demonstrate high levels of FCMR and PIGR protein in primary BCL cells using flow cytometry and immunofluorescence (IF) staining. B) Heatmap shows assays performed for each sample: healthy control (HC) CD19+ B cells; NAIVE B cells; Memory B cells (MEM); Centroblast (CB); Centrocyte (CC); CLL; DLBCL; FL.

    Article Snippet: Cells were washed with sort buffer and stained with goat anti-mouse IgG BrilliantViolet421 (BioLegend cat. 405317) and anti-CD19 APC (Miltenyi Biotec cat. 130-114-168) for 30 min. After washing, flow cytometry was performed on a modified Becton Dickinson FACScan flow cytometer and analyzed using FlowJo (v9.9.5) software.

    Techniques: Purification, Isolation, ChIP-sequencing, RNA Sequencing Assay, Microarray, Expressing, Activity Assay, Luciferase, Flow Cytometry, Immunofluorescence, Staining

    Anti-leukemic activity of CAR T cells against Ph + ALL cells Either of CAR T cells or mock T cells was co-cultured with seven different Ph + ALL cell lines at an E:T ratio of 1:5. Seven days later, co-cultured cells were stained with CD3-APC mAb and CD19-PE

    Journal: Cytotherapy

    Article Title: Anti-leukemic potency of piggyBac-mediated CD19-specific T cells against refractory Philadelphia chromosome–positive acute lymphoblastic leukemia

    doi: 10.1016/j.jcyt.2014.05.022

    Figure Lengend Snippet: Anti-leukemic activity of CAR T cells against Ph + ALL cells Either of CAR T cells or mock T cells was co-cultured with seven different Ph + ALL cell lines at an E:T ratio of 1:5. Seven days later, co-cultured cells were stained with CD3-APC mAb and CD19-PE

    Article Snippet: The expression of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) receptors on Ph+ ALL cells were assessed by means of staining with APC-conjugated CD19 mAb (Miltenyi Biotec) and PE-conjugated mAb against DR4, or DR5 (purchased from Biolegend, San Diego, CA, USA).

    Techniques: Activity Assay, Cell Culture, Staining

    CD19/CD5 positive B-CLL cells isolated from CLL1C (month 8, panel A ) and CLL5C (month 24, panel B ) patients were stained with FITC-conjugated peptide p1 or unstained. p1-positive cells corresponding to the VH 1-69 clones were 70% of total B-CLL population in CLL1C patient and 32% in CLL5C patient.

    Journal: Frontiers in Oncology

    Article Title: Predominant VH1-69 IgBCR Clones Show Higher Expression of CD5 in Heterogeneous Chronic Lymphocytic Leukemia Populations

    doi: 10.3389/fonc.2021.703254

    Figure Lengend Snippet: CD19/CD5 positive B-CLL cells isolated from CLL1C (month 8, panel A ) and CLL5C (month 24, panel B ) patients were stained with FITC-conjugated peptide p1 or unstained. p1-positive cells corresponding to the VH 1-69 clones were 70% of total B-CLL population in CLL1C patient and 32% in CLL5C patient.

    Article Snippet: Then, cells (1 × 107 cells) were first labelled with anti CD19-APC (Miltenyi Biotec, Germany, cat.n.

    Techniques: Isolation, Staining, Clone Assay