FACS was performed on an LSR II flow cytometer (BD Biosciences) and data were analyzed using FlowJo software (TreeStar). T cells were stained with the following conjugated antibodies: CD4 FITC (BD/555346), CD14 FITC (BD/555397), CD19 FITC (BD/555412), CD8 AF700 (Invitrogen/MHCD0829), murine TCR-β (mTCR-β) APC (BD/553174), and pMHC-multimers PE. Target cells with transduced WT1 or HLA-alleles were stained with: NGF-R/CD271 APC (Sanbio/CL10013APC), CD34 APC (BD/555824), murine CD19 PE (BD/557399), and HLA-A2 PE (BD/558570). Non-malignant hematopoietic subsets with: CD14 FITC (BD/555397), CD19 FITC (BD/555412), CD34 APC (BD/555824), CD80 PE (BD/557227), and CD86 PE (BD/555658). AML samples with: CD13 PE (BD/347406), CD33 FITC (BD/555626), and CD34 APC (BD/555824).
Cd19 fitc
Manufactured by BD
Sourced in United States, United Kingdom
CD19-FITC is a fluorescently labeled monoclonal antibody that specifically binds to the CD19 antigen expressed on B cells. It is designed for use in flow cytometry applications to identify and quantify B cells in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Cd3 fitc, by BD (12 mentions)
Cd4 fitc, by BD (11 mentions)
Cd14 fitc, by BD (8 mentions)
Cd4 pe, by BD (8 mentions)
Facscanto 2, by BD (7 mentions)
Cd38 pe, by BD (6 mentions)
Cd73 pe, by BD (6 mentions)
Cd14 pe, by BD (5 mentions)
Cd8 pe, by BD (5 mentions)
Facsdiva software, by BD (5 mentions)
Cd45 apc cy7, by BD (5 mentions)
Cd90 fitc, by BD (5 mentions)
Cd34 apc, by BD (4 mentions)
Cd27 apc, by Thermo Fisher Scientific (4 mentions)
Cd34 pe, by BD (4 mentions)
Cd11b pe, by BD (4 mentions)
Cd19 pe, by BD (4 mentions)
Cd56 pe, by BD (4 mentions)
Facscanto flow cytometer, by BD (4 mentions)
Cd45 fitc, by BD (4 mentions)
75 protocols using cd19 fitc
1
Multi-parameter Flow Cytometry Analysis
2
Immunophenotyping of Whole Blood Samples
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Prior to therapy, and again within 24 hours of the final therapy with IVIG, whole blood was collected in EDTA vacutainer tubes. Cyflow reagents and consumables were used according to the manufacturer’s protocol. The set comprised the following antibodies: CD4-APC/CD8-PE/CD3-FITC; CD4-APC/CD45RA-FITC/CD45RO-PE; CD19-FITC (Becton Dickinson, San Jose, CA, USA). Meanwhile, IgG1-FITC/IgG2a-PE was replied as isotype control. 100 μl of the blood was incubated in tubes together with 20 μl of the antibodies. The incubation was performed in the dark, at room temperature for 15 min. After incubation, erythrocytes were subsequently lysed, and the cell suspension was centrifuged, washed three times, and resuspended in an appropriate volume of flow staining buffer. A minimum of 10,000 cells was accepted for FACS (BD Biosciences, San Jose, CA, USA) analysis. Cells were gated based on morphological characteristics.
3
Multiparameter Flow Cytometry Analysis of Hematological Cells
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Patient-derived JMML cells or mechanically-dissociated mouse tissue cells were surface stained with monoclonal antibodies from Biolegend and Becton Dickinson: CD45-mouse Horizon Blue V605 (30-F11), CD45-human PE (HI30), CD11b AlexaFluor488 (M1/70), CD38 PerCP/Cy5.5 (HIT2), CD34 PE/Cy7 (581), CD33 APC (WM53), CD13 APC/Cy7 (WM15), CD14 Pacific Blue (M5E2), CD19 FITC (HIB19), CD66b PerCP/Cy5.5 (G10F5), CD3 PE/Cy7 (SK7), CD71 APC/Cy7 (CY1G4), CD235a BV421 (GA-R2). For intracellular staining, live cells were first stained for surface proteins and then fixed, permeabilized, and stained for intracellular proteins using the Foxp3 Transcription Factor Staining Buffer Set (Invitrogen) according to the manufacturer’s instructions. For intracellular staining, the following antibodies were used: BCL-2 PE (3F11 BD), MCL-1 PE (Y37 Abcam), BCL-XL FITC (H-5 Santa Cruz) and matching isotype controls. Normalized median fluorescence intensity (MFI) was calculated according to the following equation: normalized MFI = (MFI of protein of interest) − (MFI of each isotype). Flow cytometry was performed using a BD LSRFortessa; for analyses FlowJo software was used. The gating strategy is shown in Online Supplementary Fig. S1 .
4
Phenotyping and Sorting Plasmablasts
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PBMCs obtained from the two patients were stained with titrated amounts of CD19-FITC (BD; clone HIB19), CD3-Pacific Blue (BD; clone SP34-2), CD20-PECy7 (BD; clone L27), CD27-APC (eBiosciences; clone O323) and CD38-PE (BD; clone HIT2). The plasmablast population was defined as CD3− CD19+ CD20−/low CD27+ CD38+ lymphocytes and its frequency analyzed using FlowJo software. The single-cell sorting of plasmablasts was carried out using the FACSAriaII sorter (Becton Dickinson; Franklin Lakes, NJ, USA) at the Emory University Pediatrics Flow Cytometry Core Facility under negative air pressure. The cells were sorted into a 96-well PCR plate as described in [34 (link),35 (link)], rapidly frozen on dry ice and stored at −80 °C for subsequent cDNA synthesis.
5
Flow Cytometry Protocol for Cell Characterization
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Flow cytometry was performed as described previously46 (link). Briefly, cells were resuspended in PBS and incubated for 30 min at 4 °C with antibodies as indicated below. After staining, the cells were analyzed on a flow cytometer (BD FACS Canto II, BD Biosciences, CA, USA). The following antibodies were used: PECAM1-PE (130-092-653, Miltenyi Biotec, CA, USA), CD14-PE (555398, BD Biosciences, CA, USA), CD11b-PE (555388, BD Biosciences, CA, USA), CD34-PE (555822, BD Biosciences, CA, USA), KDR-PE (FAB357P, R&D Systems, MN, USA), CD3-FITC (555916, BD Biosciences, CA, USA), CD4-FITC (555346, BD Biosciences, CA, USA), CD19-FITC (555412, BD Biosciences, CA, USA), CD15-FITC (555401, BD Biosciences, CA, USA) and appropriate isotype control antibodies. Flow cytometric data were analyzed with FlowJo (Tree Star, Inc., Ashland, OR, USA) using appropriate isotype-matched controls.
6
Murine Hematological Characterization
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Blood collected from heart puncture was analyzed by Dept. of Veterinary Medicine hematology core at MD Anderson Cancer Center. Blood smear slides were stained with Hema-3 fixative (Fisher Healthcare) and microscopically analyzed by a hematopathologist. Splenocytes isolated from wild type, 2MX-S and 8MX-S mouse spleens were treated with RBC lysis buffer and labelled with CD19-FITC (BD Pharmingen) and CD5-PE (eBiosciences) antibodies in binding buffer. Flow analysis was carried out by flow cytometry core at MD Anderson Cancer Center.
7
Immunophenotyping of hBM-MSC with MFNP
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The immunophenotyping of hBM-MSC unlabeled and labeled with 50 µg Fe/mL of MFNP during 24 h (the maximum incubation time analyzed in this study) was performed using the following antibodies: CD19-FITC (clone:4G7) and CD34-PE (clone: 8G12) from BD Biosciences, San Jose, CA, USA; CD14-Alexa 700 (clone: M5E2), CD29-APC (clone: MAR04), CD35-FITC (clone: E11), CD44-PerCP-Cy5.5 (clone: G44-26), CD73-PE (clone: AD2), CD90-PE-Cy7 (clone: 5E10), HLA-DR-APC-H7 (clone: G46-6), CD106-FITC (clone: 51-10C9) all from BD Pharmingen, San Diego, CA, USA; CD31-V450 (clone: WM59), CD45-V500 (clone: H130), CD105-PE-CF546 (clone: 266) all from BD Horizon, San Jose, CA, USA, unstained samples and fluorescence minus one (FMO) was used as a fluorescence background control. Briefly, 1 × 105 cells were incubated in the dark/room temperature for 30 min in the presence of antibodies at concentrations recommended by the manufacturers. Cells were then washed with a buffered solution, and at least 10,000 events were acquired using FACS LSRII FORTESSA equipment (BD Biosciences). Data analyses were performed using FlowJoTM software (BD Biosciences), and results were presented as graphics where the FMO is overlaid with the stained sample for each antibody/marker used.
8
Neutrophil Identification by Flow Cytometry
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Circulating neutrophils were identified by flow cytometry using the protocol described in our previous publications [19 (link), 54 ]. In brief, 30 μl of fresh blood were incubated with fluorochrome-labelled antibody cocktail (CD19-FITC, CD16-Pacific Blue, CD11b-APC (all from BD Biosciences, Oxford, UK) and HLA-DR-PE (eBioscience, UK)) for 45 min in the dark at 4 °C. Red blood cells were removed with lysis buffer (BD Biosciences) and samples were acquired using a BD Canto II flow cytometer (BD Biosciences). The flow cytometry data were analysed using FlowJo (version 10, Tree Star Inc., Ashland, OR, USA). Neutrophils were identified based on their cell size (FSC) and granularity (SSC) as well as their cell surface antigens (CD11b+CD16+HLA-DR−).
9
Antibodies for Immunoblotting and Flow Cytometry
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Ovalbumin (OVA) grade V was purchased from Sigma-Aldrich (MO, USA). Antibodies against phospho-STAT3 (Ser727), STAT3, SOCS3, and β-actin were obtained from Cell Signaling Technology (MA, USA); anti-mouse ROCK-2 was acquired from Sta Cruz Biotech (CA, USA), and anti-mouse RhoA was purchased from Cytoskeleton (CO, USA). Flow cytometry antibodies (anti-mouse CD49d PE, CD3 PE, and CD19 FITC) were all purchased from BD (NY, USA).
10
Flow Cytometry of Immune Cells
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Flow cytometry was performed on 1E6 stained cells from each enriched fraction, including a non-enriched sample from each tonsil for comparison. Surface and intracellular flow cytometry was performed as previously described12 (link) on a Becton Dickenson LSR II flow cytometer using FACS Diva analysis software (BD Biosciences). The following commercially available antibodies were used: CD8-PE, CD19-PE, CD19-FITC, CD94-APC, CD3-APC-H7, and IFN-γ-BV421 (BD Biosciences); CD3-PerCP-Vio700, CD14-PerCP-Vio700, CD117-PE-Vio770, CD3-VioGreen, and CD14-VioGreen (Miltenyi Biotec); and IL-22-PE (R&D Systems). Cytofix/Cytoperm and PermWash reagents (BD Biosciences) were used to perform intracellular flow cytometry as previously described12 (link).
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