Immune cell phenotyping was performed under BSL 2+ conditions by incubating 200 μL of fresh whole blood in polystyrene tubes with two different fluorochrome-labeled antibody panels for 20 min in the dark [Panel 1: anti-CD3 (SP34-2, #562877), anti-CD4 (L200, #560836), anti-CD8 (SK1, #341051); anti-CD19 (HIB19, #555415), anti-CD38 (HIT2, #555460), and anti-HLA-DR (G46-6, #555811) from BD, anti-CD20 (2H7, #47-0209-42) from eBioscience, and anti-CD27 (O323, #302838) from BioLegend. Panel 2: anti-CD3 (UCHT1, #557943), anti-CD11c (O33-782, 561355), anti-CD14 (M5E2, #565283), anti-CD19 (HIB19, #557921), anti-CD123 (7G3, #554529), and anti-HLA-DR (G46-6, #560651) from BD; anti-CD16 (CB16, #47-1068) and anti-CD56 (MEM188, #17-0569) from eBioscience, and anti-CD20 (2H7, #302332) from BioLegend]. Flow cytometry was performed on an LSRII (BD) and data was analyzed using FlowJo software version 9 (Tree Star). T cells expressing both HLA-DR and CD38 were considered activated.
Anti cd19 hib19
Manufactured by BD
Anti-CD19 (HIB19) is a lab equipment product. It is a monoclonal antibody that specifically binds to the CD19 antigen, which is expressed on the surface of B cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd27 o323, by BioLegend (2 mentions)
Anti cd3 ucht1, by BD (2 mentions)
Anti cd38 hit2, by BD (2 mentions)
Anti cd3 sp34 2, by BD (1 mentions)
Anti cd4 l200, by BD (1 mentions)
Anti hla dr g46 6, by BD (1 mentions)
Anti cd8 sk1, by BD (1 mentions)
Anti cd123 7g3, by BD (1 mentions)
Ficoll density gradient centrifugation, by GE Healthcare (1 mentions)
Anti cd69 fn50, by BD (1 mentions)
Anti cd8 rpa t8, by BD (1 mentions)
Fixable live dead stain, by Thermo Fisher Scientific (1 mentions)
Anti cd107a h4a3, by BD (1 mentions)
Anti pd l1 mih1, by BD (1 mentions)
Lsrii instrument, by BD (1 mentions)
Live dead fixable red dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Human trustain fcx, by BioLegend (1 mentions)
Annexin 5, by BD (1 mentions)
Cd40 5c3, by BioLegend (1 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
7 protocols using anti cd19 hib19
1
Immune Cell Phenotyping under BSL 2+
2
Isolation and Transformation of Primary Human B Cells
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Human primary B cells isolated from adenoid from routine adenoidectomy were obtained from the Department of Otorhinolaryngology, Klinikum Grosshadern, Ludwig Maximilians University of Munich, and Dritter Orden Clinic, Munich‐Nymphenburg, Germany. All clinical samples were made fully anonymous. To isolate human primary B cells, T cells were depleted by erythrocyte rosetting using sheep red blood cells and B cells were separated by Ficoll density gradient centrifugation as recommended by the manufacturer (GE Healthcare). The remaining erythrocytes were lysed in red blood cell lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA). Cells were co‐stained by anti‐CD3+ (UCHT1; BD Pharmingen) and anti‐CD19+(HIB19; BD Pharmingen) antibodies and analyzed by flow cytometry.
To generate LCLs, primary human B cells were infected with recombinant EBV at a ratio of 1 green Raji unit (GRU) to 10 cells for 48 h and cultivated in medium containing 0.5 µg/ml cyclosporine A for 2 weeks before routine cell culture conditions were applied.
To generate LCLs, primary human B cells were infected with recombinant EBV at a ratio of 1 green Raji unit (GRU) to 10 cells for 48 h and cultivated in medium containing 0.5 µg/ml cyclosporine A for 2 weeks before routine cell culture conditions were applied.
3
Multiparametric Immune Profiling
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Fixable Live/Dead stains (Invitrogen) were utilized according to manufacturer’s instructions prior to surface staining. Following quenching and washing, surface staining was performed at 4 °C in RPMI media +1% FBS unless otherwise indicated. Antibody clones (manufacturer) utilized in this study: anti-CD8: RPA-T8 (BD), anti-CD107a: H4A3 (BD), anti-PD-1: EH12.2H7 (eBioscience), anti-PD-L1: MIH1 (BD), anti-CD69: FN50 (BD), anti-β2M: TU99 (BD), anti-CD19: HIB19 (BD). Data were collected on an LSR II (BD) and analyzed using FlowJo.
4
Lymphocyte Subsets Pharmacodynamics
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The PD variables were absolute lymphocyte count (ALC), absolute T‐cell count, and absolute B‐cell count, assessed at screening, on day −1, and at 0, 72, 168, 240, 336, 504, 672, and 1,344 hours postdose. Absolute T‐cell and B‐cell counts were ALCs multiplied by the proportions of CD3+ and CD19+ cells against total lymphocytes, respectively, determined using multicolor flow cytometry. Cryopreserved peripheral blood mononuclear cells were thawed and stained using the LIVE/DEAD Fixable Red Dead Cell Stain Kit (Life Technologies, Waltham, MA). The cells were then washed once and stained with anti‐CD3 (UCTH1; BD Biosciences, San Jose, CA) and anti‐CD19 (HIB19; BD Biosciences) in the dark at 4°C for 30 minutes. Multicolor flow cytometry was performed on an LSR II instrument (BD Biosciences) and data were analyzed using FlowJo software (Treestar, Ashland, OR).
For the PD variables, the percent change from baselines was calculated, where the baseline value was the average of those measured at screening, on day –1, and at 0 hour postdose on day 1. Furthermore, the maximum ALC and the area under the ALC‐time curve until the last sampling time were calculated using the Phoenix WinNonlin software after correcting the baseline values.
For the PD variables, the percent change from baselines was calculated, where the baseline value was the average of those measured at screening, on day –1, and at 0 hour postdose on day 1. Furthermore, the maximum ALC and the area under the ALC‐time curve until the last sampling time were calculated using the Phoenix WinNonlin software after correcting the baseline values.
5
Characterization of Dendritic Cell Surface Markers
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DCs were harvested 20 h poststimulation by pipetting the cells up and down. Cell death was assessed using the LIVE/DEAD fixable aqua dead cell stain kit (Life Technologies, Inc., Carlsbad, CA) as well as annexin V (BD Biosciences, San Jose, CA). Fc receptor blocking was performed with human TruStain FcX (BioLegend, San Diego, CA). DCs were stained with fluorochrome-conjugated antibodies against surface markers for 20 min on ice. The antibodies used were directed against CD3 (catalog number HIT3α), CD11c (3.9), HLA-DR (L243), CD86 (IT2.2), CD83 (HB15e), CD80 (2D10), and CD40 (5C3) and were all purchased from BioLegend (San Diego, CA). Anti-CD14 (M5E2) was obtained from eBioscience (San Diego, CA), and anti-CD19 (HIB19) was from BD Biosciences (San Jose, CA). DCs were then washed, samples were acquired on a BD LSR II apparatus, and data were analyzed using FlowJo software (version 9.9.3). DCs analyzed for costimulatory molecule expression were gated on the live, CD3/CD19-negative, CD11c-positive population.
6
Multiparametric Flow Cytometry Immune Profiling
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PBMCs were isolated using Ficoll-Paque density gradient centrifugation, frozen in freezing medium [10% DMSO, 90% fetal bovine serum (FBS)] and stored at -80°C until use. For staining, PBMCs were thawed using complete RPMI (RPMI+10%FBS) and washed with PBS. The cells were first stained using Fixable Aqua Dead Cell Kit (Thermofisher), followed by staining with anti-CD3 (UCHT1) (Biolegend), anti-CD4 (RPA-T4) (BD Biosciences), anti-CD8 (SK1) (Biolegend), anti-CD19 (HIB19) (BD Biosciences), anti-CD27 (O323) (Biolegend), anti-CD56 (B159) (BD Biosciences), anti-CD45RA (2H4) (Beckman Coulter), anti-CD38 (HIT2) (BD Biosciences) and anti-CD38 (JK36) (Beckman Coulter). The cells were washed and analyzed using BD LSRFortessa™ cell analyzer. Data analyses were performed using Flowjo V10.5.3 (BD)
7
Phenotypic Analysis of PBMCs by Flow Cytometry
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Cell staining and the flow cytometry‐based phenotypic analyses of PBMCs and cells were performed according to standard flow cytometry methods (Martin et al, 2014 ; Izawa et al, 2017 ). The following monoclonal antibodies were conjugated to phycoerythrin‐cyanin7 (PE‐Cy7) Brilliant Violet 785 (BV785), Brilliant Violet 510 (BV510), Brilliant Violet (BV650), phycoerythrin (PE), phycoerythrin‐cyanin5 (PE‐Cy5), Brilliant Violet 451 (BV421), Peridinin‐chlorophyll‐cyanin5.5 (PerCP‐Cy5.5): anti‐CD25 (BC96; dilution 1:40), anti‐CD3 (OKT3; dilution 1:40), anti‐CD4 (OKT4; dilution 1:40), anti CD8 (RPA‐T8; dilution 1:40), anti‐CD27 (O323; dilution 1:40), anti‐CD45RA (HI100; dilution 1:40), anti‐CD161 (HP‐3G10; dilution 1:20), anti‐TCR Vα7.2 (3C10; dilution 1:20) all purchased from Sony Biotechnology Inc.; anti‐TCR Vα24 (C15; dilution 1:20), anti‐TCR Vβ11 (C21; dilution 1:20), anti‐TCR γδ (IMMU510; dilution 1:40) from Beckman Coulter; and anti‐CD19 (HIB19; dilution 1:40) and anti‐CD57 (NK‐1; dilution 1:200) from BD Biosciences. For intracellular staining of CTPS1, cells were fixed and permeabilized using the Intraprep kit (Beckman Coulter) according to the manufacturer's instructions. Cells were stained with an anti‐CTPS1 antibody (EPR8086B, Abcam; dilution 1:200) or an isotype‐matched antibody and then labeled with a FITC‐goat anti‐rabbit secondary antibody.
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