Lymphocyte subpopulations were measured at baseline of each exercise, at iso-workload and at peak of each exercise. Lymphocyte subpopulations were not measured at 3 and 6 h after each exercise. To evaluate the percentages of lymphocyte, NK, T and B lymphocyte, 2 × 10 5 cells were collected and stained with anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD16, anti-CD20 or anti-CD56 fluorochrome-conjugated MAbs (Becton Dickinson). Cells were analysed on a FACSCalibur cytofluorimeter, using the CellQuest software (Becton Dickinson). The area of positivity was determined by use of an isotype-matched MAb.
Anti cd20
Manufactured by BD
Sourced in United Kingdom, Germany
Anti-CD20 is a monoclonal antibody that binds to the CD20 antigen expressed on the surface of B cells. It is a laboratory tool used in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd3, by BD (6 mentions)
Anti cd8, by BD (3 mentions)
Anti cd27, by BD (3 mentions)
Cellquest software, by BD (2 mentions)
Anti cd23, by BD (2 mentions)
Anti cd38, by BD (2 mentions)
Anti cd4, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Pe conjugated anti cd19, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Lsrfortessa cell analyser, by BD (1 mentions)
Easysep human b cell enrichment kit, by STEMCELL (1 mentions)
Anti cd5, by BioLegend (1 mentions)
Cytexpert v2, by Beckman Coulter (1 mentions)
Cytoflex s, by Beckman Coulter (1 mentions)
Facsdiva software, by BD (1 mentions)
Anti cd45, by BD (1 mentions)
Anti cd19, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Igg isotype control, by BD (1 mentions)
10 protocols using anti cd20
1
Lymphocyte Subpopulation Analysis
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
Immunophenotyping of B and T cells
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Fluorochrome-conjugated mAbs anti-CD3 [phycoerythrin (PE)-Cy7], anti-CD19 (Alexa Fluor 700), anti-CD45 (PE), anti-CD20 (FITC) and anti-CD32 (PE) were obtained from BD Biosciences (Oxford, UK) and Biolegend, London, UK. In addition to forward- and side-scatter characteristics, B cells were identified as CD19+ and T cells as CD3+, by flow cytometry using a Becton Dickinson LSR Fortessa cell analyser (supplementary Figs S1 and S2 , available at Rheumatology online). Peripheral blood mononuclear cells were separated from whole blood by Ficoll-Hypaque density gradient and B cells were isolated using EasySep Human B Cell Enrichment Kit (Stemcell Technologies, Cambridge, UK).
4
Isolation and Characterization of B Cells from Peripheral Blood
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PB samples were obtained after written informed consent was received from participants prior to inclusion in the study according to the Declaration of Helsinki, and approval by the ethics committee of the Medical Faculty at the University of Duisburg‐Essen, Germany (BO‐10‐4380). B cells were isolated by Ficoll density centrifugation (density 1.077 g/ml, Pan BioTech, Aidenbach, Germany) followed by staining with anti‐CD3 (BD Biosciences, Heidelberg, Germany), anti‐CD5 (BioLegend, Koblenz, Germany), anti‐CD20, anti‐CD23, and anti‐CD27 (each BD Biosciences) antibodies. Stained cells were analyzed on a CytoFLEX S flow cytometer (Beckman Coulter, Krefeld, Germany) using CytExpert v2.4 (Beckman Coulter) or FlowJo v10.6.2 (BD Biosciences) software. RNAseq data were retrieved from (Budeus et al, 2021 (link)).
5
Cynomolgus Monkey Lymphocyte Immunophenotyping
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Peripheral blood samples from cynomolgus monkeys were stained with saturating concentrations of fluorescently conjugated mAbs to human CD3, CD8, CD20 and CD45 (all cross‐react with cynomolgus monkey leukocytes), and with appropriate isotype controls. Samples were incubated on ice, washed twice with FACS staining buffer and re‐suspended in fixative buffer before acquisition. Flow cytometric acquisition was performed on the BD FACSCantoTM II on the day of staining. Data were analysed with BD FACSDivaTM software, version 6.1.1 (BD Biosciences). Flow cytograms were generated to establish the fraction of cells expressing each cell surface marker (expressed as percentage of FSC/SSC‐gated lymphocytes). B cells were defined as CD3−CD20+ cells, T‐cells as CD3+ cells (cytotoxic T‐cells as CD3+CD8+ cells, helper T‐cells as CD3+CD8− cells), and NK cells as CD3−CD20− cells. For each sample at each time point for each lymphocyte subset, the absolute cell count was calculated based on % gated data generated by flow cytometry multiplied by the total lymphocyte count. Statistical analysis was not performed due to the low number of animals assigned to each dose group. All antibodies (anti‐CD3, anti‐CD8, anti‐CD20 and anti‐CD45) were purchased from BD Biosciences.
6
Plasmablast Identification in PBMCs
7
Immunophenotyping of RT-DLBCL cells
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To determine the immunophenotype of the RT-DLBCL cells, HPRT3, HPRT2, and HPRT1 cells were harvested from NSG mice. Cells were suspended in 100 µL of 0.5% BSA/PBS and stained with fluorophore-conjugated anti-CD19, anti-CD5, anti-CD10, anti-CD20, anti-CD23, anti-PD-1 or IgG-isotype controls (BD Biosciences, San Jose, CA). Percent expression of each cell surface marker is reported relative to the respective IgG isotype control.
8
Isolation and Co-Culture of Tumor-Infiltrating Cells
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The mononuclear cells among tumors were separated using the Ficoll-Paque PLUS density gradient media (GE health). The pellets of dead cells were filtered with the 40-μm Cell Strainer (BD Falcon). And the mononuclear cells were stained with anti-BCMA (357503, Biolegend) or anti-CD20 (555622, BD Biosciences) antibodies. BCMA-positive or CD20-positive cells were sorted with the BD FACSAria II. The sorted cells were cultured in RPMI 1640 Medium supplemented with 10% FBS and 200 U/mL penicillin-streptomycin. After being sorted for 24 h, cells were centrifuged and resuspended in fresh RPMI 1640 Medium supplemented with 10% FBS. The supernatant of sorted cells was collected 48 h after culture. For co-culture experiments, the sorted cells were co-cultured with A549 and H1299 cells in DMEM supplemented with 10% FBS for 48 h. After 48 h of co-culture, B cell-containing culture media were removed and replaced with fresh culture media and cultured for further 4 h, and the cell viability of A549 and H1299 cells was determined by CCK8 assays.
9
Characterization of MDM2, p73, and Ubiquitin Interactions
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GST-MDM2, p21-Luc, pCMV-Bam-MDM2, and MDM2ΔRING have been described previously [20 (link), 21 ]. Myc-Mdm2 was kindly provided by Dr. Jochemsen. All Ub and Ub mutants were PCR-amplified and subcloned into pET28a. The mouse Mdm2 was also cloned into pcDNA3 and confirmed by sequence. Flag-p73α, p73β, and p73 mutants were generated by PCR and subcloned into pCMV-Tag1 (Stratagene). p73α and p73β were also cloned into pcDNA3 without tag. All PCR products were confirmed by sequencing. Anti-p73 (Ab-1 and Ab3, Oncogene Science; H-79, Santa Cruz Biotechnology; ab40658, Abcam), anti-MDM2 (2A10, Calbiochem; SMP14, Santa Cruz Biotechnology; MD-219, Sigma), anti-Myc (9E10, Roche), anti-Flag (M5, Sigma), anti-GFP (B-2, Santa Cruz Biotechnology), anti-GST (B-14, Santa Cruz Biotechnology), anti-HA (12CA5, Roche), anti-ubiquitin (BD Bioscience), anti-actin (Sigma), anti-CD20 (Pharmingen), and polyubiquitin-specific FK-1, anti-ubiquitin, Lys63-specific and Lys48-specific antibodies (Millipore) were used according to the manufacturers’ instructions.
10
Generation of DENV-specific Human mAbs
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