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
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
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
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
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
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
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
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
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