Primary CLL cells were incubated for 24 hours with a physiological dose of 25 μM bendamustine, kindly provided by Mundipharma. When indicated, CLL cells were pretreated for 24 hours with 0.5 μM of the PI3Kδ inhibitor idelalisib and 1 μM of the BTK inhibitor ibrutinib (Selleck Chemicals) prior to bendamustine addition. Cell viability was quantified by double staining with Annexin-V conjugated to fluorescein isothiocyanate (FITC) and PI (eBiosciences). For the comparative analysis of response in PB, BM and LN compartments, cells were triple-stained with phycoerythrin (PE)-conjugated anti-CD19 (Becton Dickinson), Annexin-V-FITC and PI. Labeled samples were analyzed on an Attune focusing acoustic cytometer (Life Technologies). Cytotoxicity (mean ± SEM) was calculated as the percentage of Annexin-V-positive cells in treated samples relative to the untreated ones.
Pe conjugated anti cd19
Manufactured by BD
Sourced in United States
PE-conjugated anti-CD19 is a fluorescent-labeled antibody that binds to the CD19 cell surface antigen. It is commonly used in flow cytometry applications to identify and quantify CD19-positive cells.
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Lab products found in correlation
Facscanto, by BD (3 mentions)
Fitc conjugated anti cd3, by BD (2 mentions)
Idelalisib, by Selleck Chemicals (1 mentions)
Ibrutinib, by Selleck Chemicals (1 mentions)
Attune acoustic focusing cytometer, by Thermo Fisher Scientific (1 mentions)
Anti cd20, by BD (1 mentions)
Anti cd8, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Calibur flow cytometer, by BD (1 mentions)
Pe cy7 conjugated anti cd25, by BD (1 mentions)
Pe conjugated anti ly6g, by BD (1 mentions)
Percp conjugated anti cd3, by BD (1 mentions)
Pe conjugated anti cd8, by BD (1 mentions)
Bv421 conjugated anti cd4, by BD (1 mentions)
Apc conjugated anti cd206, by BD (1 mentions)
Fitc conjugated anti cd49b, by BD (1 mentions)
Apc conjugated anti nkp46, by BD (1 mentions)
Pe cy7 labeled streptavidin, by BD (1 mentions)
Apc conjugated anti foxp3 antibody, by Thermo Fisher Scientific (1 mentions)
12 protocols using pe conjugated anti cd19
1
Assessing Bendamustine Cytotoxicity in CLL
2
Flow Cytometric Analysis of HLA-DR Expression
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The surface and cytoplasmic expressions of HLA-DR in different lymphocyte subpopulations were compared as follows. PBMCs were stained for cell-surface antigens before cell membrane permeabilization using FITC-conjugated anti-CD4, anti-CD8, anti-CD20, or anti-CD56 mAb (Becton Dickinson). After washing, the cells were fixed and permeabilized with a Cytofix/Cytoperm Plus kit (Becton Dickinson) and then incubated with PE-conjugated anti-HLA-DR at 4 °C for 20 min. Stained cells were analyzed with a FACSCalibur flow cytometer using CellQuest software (BD Bioscience, Tokyo, Japan). After gating FITC-positive cell clusters for each lymphocyte subpopulation, the HLA-DR expression profiles were compared between the surface and cytoplasmic expressions.
The expression of HLA class II molecules was examined in a similar manner. For HLA-DR, HLA-DQ, and CLIP expressions, FITCconjugated antibodies were used together with PE-conjugated anti-CD19 (Becton Dickinson). For HLA-DM, PE-conjugated antibody was used together with FITC-conjugated anti-CD19 (Becton Dickinson). The mean fluorescence intensity (MFI) of each sample was determined by flow cytometry after gating CD19 + B cells. The differences between the surface and cytoplasmic staining intensities were calculated and the data are presented as the ΔMFI.
The expression of HLA class II molecules was examined in a similar manner. For HLA-DR, HLA-DQ, and CLIP expressions, FITCconjugated antibodies were used together with PE-conjugated anti-CD19 (Becton Dickinson). For HLA-DM, PE-conjugated antibody was used together with FITC-conjugated anti-CD19 (Becton Dickinson). The mean fluorescence intensity (MFI) of each sample was determined by flow cytometry after gating CD19 + B cells. The differences between the surface and cytoplasmic staining intensities were calculated and the data are presented as the ΔMFI.
3
Spleen Biopsy for B Cell Monitoring in ABOi LDLT
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We performed spleen biopsy during ABOi LDLT desensitization with rituximab in all patients, except in patients who underwent splenectomy before transplantation. Our objective was to validate the elimination of B cells from the spleen and determine if the patient required splenectomy. The cells were stained with fluorescein isothiocyanate-conjugated anti-IgM (BD Pharmingen) and PE-conjugated anti-CD19 (BD Pharmingen) and analyzed using a fluorescence-activated cell sorter Calibur flow cytometer (Becton Dickinson). The rate of residual B cells in splenocytes and peripheral blood mononuclear cells was determined by selecting cells gated on IgM+ and CD19+ lymphocytes.
We performed splenectomy only if B cells were clearly detected in the spleen by flow cytometry analyses of the biopsy specimens at the time of transplantation (ie, in cases where more than 1% of IgM+ CD19+ cells were detected among the splenocytes).
We performed splenectomy only if B cells were clearly detected in the spleen by flow cytometry analyses of the biopsy specimens at the time of transplantation (ie, in cases where more than 1% of IgM+ CD19+ cells were detected among the splenocytes).
4
Comprehensive Immune Cell Phenotyping by Flow Cytometry
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For surface marker staining, cells were stained in PBS supplemented with 1% FBS for 30 min on ice with the following antibodies (PerCP‐conjugated anti‐CD3, BV421‐conjugated anti‐CD4, PE‐conjugated anti‐CD8, BV570‐conjugated anti‐CD45, BV421‐conjugated anti‐F4/80, BV650‐conjugated anti‐CD86, APC‐conjugated anti‐CD206, PE‐Cy7‐conjugated anti‐CD25, FITC‐conjugated anti‐γδ TCR, APC‐Cy7‐conjugated anti‐CD11b, PE‐conjugated anti‐Ly‐6G, FITC‐conjugated anti‐CD49b, PE‐conjugated anti‐CD19, PE‐conjugated anti‐ST2 antibodies, and APC‐conjugated anti‐NKp46: BD Biosciences, San Jose, CA, USA). After fixation and permeabilization, staining with an APC‐conjugated anti‐FOXP3 antibody (eBioscience) was performed according to the manufacturer's instructions. For IL‐33 staining of primary mouse hepatocytes, cells were stained using a biotinylated anti‐IL‐33 monoclonal antibody (Enzo Life Biosciences, Raamsdonksveer, The Netherlands) and PE‐Cy7‐labeled streptavidin (BD Pharmingen, San Diego, CA, USA). All the flow cytometric data were analyzed and plotted using FlowJo software (TreeStar, Ashland, OR, USA).
5
Isolation and Characterization of Plasma Cells
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Bone marrow mononuclear cells were isolated on Ficoll-Hypaque gradient and plasma cells were isolated using CD138 magnetic beads (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to manufacturer’s instructions. Purity was assessed by flow cytometry (FACS Canto, Becton Dickinson) and the following labeled mAbs: FITC-conjugated anti-CD38, PE-Conjugated anti-CD138, PE-Conjugated anti-CD19, Cychrome-Conjugated anti-CD45 and APC-H7-conjougated CD56 (BD Biosciences, Le Pont de Claix, France). Purity of isolated plasma cells (CD38+ CD138+) ranged from 95% to 99%.
6
Comprehensive Immune Cell Profiling
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The V450-conjugated anti-CD3, Alexa Fluor®700-conjugated anti-CD3, BV650-conjugated anti-CD4, FITC-conjugated anti-CD4, BV605-conjugated anti-CD11b, BV421-conjugated anti-CD11c, BV650-conjugated anti-CD14, PE-CyTM7-conjugated anti-CD14, Alexa Fluor®700-conjugated anti-CD16, BV421-conjugated anti-CD16, PE-CF594-conjugated anti-CD16, phycoerythrin (PE)-conjugated anti-CD19, Alexa Fluor®700-conjugated anti-CD19, Alexa Fluor®700-conjugated anti-CD20, V500-conjugated anti-CD45, BV786-conjugated anti-NKp46, Alexa Fluor®700-conjugated anti-CD56, PE-conjugated anti-CD64, PE-CyTM7-conjugated anti-CD86, PerCP-Cy5.5-conjugated anti-CD123, BV711-conjugated anti-CD141, BV786-conjugated anti-PDL2, and PE-CF594-conjugated anti-CD163 antibodies were from BD Biosciences. The APC-Alexa Fluor®750-conjugated anti-HLA-DR antibody was purchased from Beckman Coulter. The PE-conjugated anti-CD1c, APC-conjugated anti-CD303, and APC-conjugated anti-SLAN antibodies were from Miltenyi Biotech. The FITC-conjugated anti-CD192 antibody was purchased from R&D Systems. All antibodies were used according to their manufacturer's recommendations. Antibodies combination for monocytes: CD3, CD11b, CD14, CD16, CD19, CD45, CD56, CD64, CD163, CD192, NKp46, HLA-DR, SLAN, and DC: CD1c, CD3, CD4, CD11b, CD11c, CD14, CD16, CD19, CD20, CD45, CD56, CD86, CD123, CD141, CD303, PDL2, HLA-DR.
7
Flow Cytometry Analysis of B Cell Depletion
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Laboratory analysis of B cell depletion was done using flow cytometry at baseline (before the infusion) and at 2 and 24 weeks post-infusion. A peripheral blood sample was collected, and phycoerythrin (PE)-conjugated anti-CD19 (BD Biosciences-US) was added and incubated for 20 min, followed by red blood cell lysis and washing in phosphate-buffered saline. The cells were then counted in a BD FACS Canto flow cytometer. A minimum of 20 000 events were acquired and analysed with BD FACS Canto clinical software, and the CD19 percentage was determined. A percentage of B cells in the peripheral blood of <0.01% was considered as complete depletion.
8
ADSC Molecular Characterization by Flow Cytometry
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For molecular characterization, ADSCs were stained with a PerCP-Cy-conjugated anti-CD73 (Cat. # 561260, BD Pharmingen), APC-conjugated anti-CD90/Thy1 (Cat. # 559869, BD Pharmingen), PE-conjugated anti-CD11b (Cat. # 557321, BD Pharmingen), and PE-conjugated anti-CD19 (Cat. # 561741, BD Pharmingen) antibodies purchased from BD Bioscience (BD Biosciences, San Jose, CA, USA) and diluted at 1:125. Data were analyzed with the BD FACSDiva software v. 8.0.1 using a BD LSRFortessa cell analyzer (BD Biosciences, San Jose, CA, USA).
9
Isolating and Analyzing Bone Marrow Leukocytes
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Bone marrow cells were harvested from femur using a syringe with 5 ml of PBS. Pelleted cells were resuspended in Tris-buffered 0.83% NH4Cl solution to lyse erythrocytes, washed in PBS and resuspended in FACS buffer (PBS supplemented with 10% FCS (Sigma) and 0.1% NaN3). The total number of leukocytes was counted using Nucleocassettes and Nucleocounter (Chemometec). Cells were stained with PE-conjugated anti-CD19 (BD) and analyzed using a FACSVerse (Becton Dickinson). FlowJo software version 7.6.5 (Tree Star, Ashland, USA) was used for data analysis.
10
Multicolor Flow Cytometry Panel
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The following antibodies against hu-Ag were used: FITC-conjugated anti-CD3, -CD14, and -CD62L, PEconjugated anti-CD19, -CD16, -CD56, -CD45RA, -CCR4, and -CCR6, PerCP-Cy5.5-conjugated anti-HLA-DR, PE-Cy7-conjugated anti-CD3, APC-conjugated anti-CD3, -CD45, and -CCR6, Alexa-Fluor 647-conjugated anti-CD127, Alexa-Fluor 488-conjugated anti-Foxp3, APC-H7-conjugated anti-CD8 and -CD45RO, BV421conjugated anti-CD25 and -CXCR3, and V500-conjugated anti-CD4 (all from BD Pharmingen, San Diego, USA). Dead cells were excluded by 7AAD staining. For intracellular staining, Fixation/Permeabilization solution (eBioscience, San Diego, USA) was used. Multicolor cytometric analysis was performed on a FACS Canto II (BD Bioscience, New Jersey, USA) and data were analyzed by FlowJo software (FLOWJO LLC).
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