In contrast to the other effector cell assays, B cells were co-cultured directly with freshly isolated DCs. Autologous B cells were incubated with or without DCs at a 10:1 ratio (B cell:DC) in the presence of different stimuli, for 7 d at 37°C. DCs were CD1c+ DCs, pDCs or a combination of both at a 1:1 ratio, with each well containing equal total DC numbers. After 7 d, B cells were stained with CD20-APC (eBioscience, 17-0209-42) and CD38-PE (ImmunoTools, 21270384) or CD38-PerCP-Cy5.5 (BD Biosciences, 551400) and a viability dye (Fixable Viability Dye eFluor 780 or 506; eBioscience, 65-0865-18 or 65-0866-18) and measured on a FACSVerse. Plasma cells were defined as CD38highCD20low expressing cells.
Cd38 percp cy5
Manufactured by BD
Sourced in United States
CD38-PerCP-Cy5.5 is a fluorescently-labeled monoclonal antibody that binds to the CD38 antigen. It is designed for use in flow cytometry applications to identify and quantify CD38-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd123 pe, by BD (3 mentions)
Il 21, by Thermo Fisher Scientific (2 mentions)
Cd16 apc h7, by BD (2 mentions)
Cd56 pe cy7, by BD (2 mentions)
Cd8 v500, by BD (2 mentions)
Anti human cd3 apc h7, by BD (2 mentions)
Live dead fixable dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Cd4 pe cy7, by BD (2 mentions)
Hla dr fitc, by BD (2 mentions)
Cd24 pe, by BD (2 mentions)
Act diff hematology analyzer, by Beckman Coulter (2 mentions)
Cd45ra fitc, by Beckman Coulter (2 mentions)
Cd34 apc, by Agilent Technologies (2 mentions)
Histopaque 1077, by Merck Group (2 mentions)
Igd fitc, by BD (2 mentions)
Cd20 apc, by Thermo Fisher Scientific (1 mentions)
Fixable viability dye efluor 506 or 780, by Thermo Fisher Scientific (1 mentions)
Anti glyceraldehyde 3 phosphate dehydrogenase gapdh, by Santa Cruz Biotechnology (1 mentions)
Bca protein reagent, by Thermo Fisher Scientific (1 mentions)
Recombinant human cxcl12, by Thermo Fisher Scientific (1 mentions)
14 protocols using cd38 percp cy5
1
Quantification of Plasma Cell Differentiation
2
Immune Cell Activation by CXCL12 and IL-21
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Recombinant human CXCL12 and IL-21 were purchased from Peprotech (Rocky Hill, NJ). Peptidoglycan (PGN) poly (I:C), LPS, and R848 were purchased from Sigma-Aldrich (Poole, Dorset, UK). Human CpG-B DNA was purchased from Hycult Biotech (Uden, Netherlands). Flagellin was provided by Dr. Myoung Ho Jang (Osaka University). RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Gibco BRL (Eggenstein, Germany). Anti-ERK1/2 and anti-human phospho-AKT1/2/3 monoclonal antibodies (Ab) were obtained from Cell Signaling Technology. Anti-ß-actin and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Abs were obtained from Santa Cruz (Paso Robles, CA). Anti-RGS1 Ab was purchased from Novus Biologicals (Littleton, CO). The anti-CXC chemokine receptors (CXCR) 4-APC, TCRαβ-FITC, CD38-PerCP-Cy5.5, and CD20-APC were purchased from BD Biosciences (Heidelberg, Germany). The BCA protein reagent was purchased from Thermo Scientific (Hudson, NH). Interleukin (IL)-2 and IL-4 were provided by Prof. Jongseon Choe (College of Medicine, Kangwon National University).
3
Isolation and Phenotypic Analysis of Naïve B Cells
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PBMCs were isolated from whole blood collected from family members and healthy donors by ficoll-histopaque gradient centrifugation. For phenotypic staining, the following monoclonal antibodies were used: CD3-APC-H7, CD4-PerCP-Cy5.5, CD38-PerCP-Cy5.5, CD10-PECF594, CD21-APC, IgG PeCy7, CD14-PerCP, CD123-PE, CD56-PeCy7, CD11c-APC, CD16-APC-H7 (BD Biosciences, San Diego, CA, USA), CD8-APC-EF780, CD27-APC-EF780 (eBioscience, San Diego, CA, USA), CD19-BV650, CD24-BV605 (Biolegend, San Diego, CA, USA) and IgA-PE (Miltenyi Biotech, Bergisch Gladbach, Germany). Naïve B cells were enriched by negative selection using B-cell isolation kit (Stemcell, Vancouver, BC, Canada). Naïve B-cell purity was verified by flow cytometry to 98% purity.
4
Comprehensive Multicolor Flow Cytometry for Plasma Cell Analysis
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Abs used were CD19 PE (LT19; Miltenyi Biotec), CD138 allophycocyanin (B-B4; Miltenyi Biotec), CD38 PE-Cy7 (HB7; BD Biosciences), CD38 AF700 (HIT2; BioLegend), CD20 eFluor V450 (2H7; eBioscience); CD27 AF647 (LT27; AbD Serotec), CD27 FITC (M-T271), CD19 PerCP-Cy5.5 (SJ225C1; BD Biosciences), CD19 PE-Cy7 (SJ225C1; BD Biosciences), CD24 FITC (ML5; BD Biosciences), CD84 PE (CD84.1.21; BioLegend), CD38 PerCP-Cy5.5 (HIT2; BD Biosciences), CD95 BV421 (DX2; BioLegend), CD20 allophycocyanin-H7 (L27; BD Biosciences), CD27 BV605 (O323; BioLegend), CD3 VioGreen (BW264/56; Miltenyi Biotec), Ki67 FITC (B56; BD Biosciences), unconjugated goat anti-IRF4 (M-17; Santa Cruz Biotechnology), and donkey anti-goat IgG AF488 (polyclonal; Invitrogen). Controls were isotype-matched mouse mAbs. Annexin V FITC was from eBioscience, and 7-AAD was from BD Biosciences.
Reagents included human IL-2 (Roche), IL-6 (PeproTech), IFN-α (Sigma), IL-21 (PeproTech), goat anti-human F(ab′)2 fragments (anti-IgM and anti-IgG; Jackson ImmunoResearch), HybridoMax hybridoma growth supplement (Gentaur), Lipid Mixture 1, Chemically Defined (200×), and MEM Amino Acids Solution (50×, Sigma).
Reagents included human IL-2 (Roche), IL-6 (PeproTech), IFN-α (Sigma), IL-21 (PeproTech), goat anti-human F(ab′)2 fragments (anti-IgM and anti-IgG; Jackson ImmunoResearch), HybridoMax hybridoma growth supplement (Gentaur), Lipid Mixture 1, Chemically Defined (200×), and MEM Amino Acids Solution (50×, Sigma).
5
T Cell Activation Profiling of Healthy Volunteers
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PBMC from 20 healthy volunteers were isolated from whole peripheral blood using a density gradient (LymphoprepTM, AXIS-SHIELD) centrifugation and frozen until use. Briefly, PBMC were cultured for 3 days in RPMI 1640 media supplemented with 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin and 20% fetal calf serum (GibcoTM, Life Technologies). For activation, 5 μg/ml PHA (Sigma-Aldrich) and 50 U/ml of recombinant human IL-2 (Roche, Sigma-Aldrich) were added to the media. Viability staining (Live/Dead Fixable Dead Cell Stain kit, Invitrogen) was performed to gate on viable cells and T cell activation was assessed by staining for T cell markers (antibodies: anti-human CD3-APC-H7, CD4-PE-Cy7, and CD8-V500; BD Biosciences) and markers of T cell activation (antibodies: anti-human CD25-APC, HLA-DR-FITC and CD38-PerCP-Cy5.5; BD Biosciences). Cells were collected on an LSR II instrument (Becton Dickinson) and T cell activation markers analysis was performed using the FlowJo software.
6
Peripheral Blood Lymphocyte Immunophenotyping
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Immunophenotyping of peripheral blood lymphocyte subsets were mainly based on Reference [31 (link)]. Briefly, EDTA-anticoagulated whole blood was split into 2 parts and stained with different panels of antibodies to explore T cells and B cells separately. For T cell subsets, the antibody panel contained CD3-PerCP-Cy5.5, CD4-FITC, CD8-BV510, CD45RA-PE-Cy7, CD27-APC, TCR aβ-PE, and TCR γδ-BV421. For B cell subsets, the antibody panel contained CD19-APC, CD27-V450, IgD-BV510, CD24-PE, and CD38-PerCP-Cy5.5 (All antibodies were purchased from BD Biosciences). After 20 min, erythrocytes in blood sample were lysed by red cell lysis buffer (TIANGEN, China). Then cells were centrifuged and washed twice with PBS. After resuspended, the cells were acquired on BD FACS Canto II, and the data were analyzed using FlowJo (V10.4, BD Biosciences).
7
Purification of Human Hematopoietic Stem Cells
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Mobilised peripheral blood samples were thawed, washed in PBS and incubated at 37°C with PBS/1% FBS/1 mM EDTA (FACS Buffer) for 30 mins and non-adherent cells collected to remove monocytes. Lineage-negative cells were collected by using EasySep® Human Progenitor Cell Enrichment Kit (STEMCELL Technologies, Victoria, Australia). Cells were incubated with EasySep® Enrichment Cocktail followed by EasySep® Magnetic Nanoparticles. Cells were then incubated for 10 mins in the EasySep® magnet, where lineage-negative cells were magnetically separated. Lineage-negative cells were stained with the following fluorophore-conjugated antibodies: Lineage-FITC, CD34-phycoerythrin (PE)Cy7, CD38-PerCP-Cy5.5, CD45RA-PacificBlue, CD90-APC, CD123-PE (BD Biosciences, San Jose, CA, USA). Cells were FACS sorted and analyzed using BD Influx Cell Sorter (BD Biosciences, San Jose, CA, USA). Cells were collected in sterile FACS buffer, and stored at -80°C in QIAzol (Qiagen, Valencia, CA, USA) until RNA extraction.
8
Monitoring T Cell Activation and HIV Co-Receptors
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Benzyl-2-acetamido-2-deoxy-α-d -galactopyranoside-treated and -untreated PHA-blasts were used to monitor expression of surface markers of cell activation and HIV co-receptor CCR5 and CXCR4. Briefly, PHA-blasts were prepared from 20 HIV-negative donors by stimulation with PHA for 2 days. One-half of the cells from each sample was then treated with 2 mM of BAGN overnight. The next day, cells were stained for viability markers (Live/Dead Fixable Dead Cell Stain kit, Invitrogen) and markers of T cell activation (antibodies: anti-human CD3-APC-H7, CD4-PE-Cy7, CD8-V500, CD25-APC, HLA-DR-FITC, and CD38-PerCP-Cy5.5; BD Biosciences). Cells were fixed and permeabilized (Fix and Perm, Invitrogen) to stain with Ki67-PE (BD Biosciences). Additional aliquots of BAGN-treated and -untreated PHA-blasts were used to stain for viability and T cell subsets as before and for HIV co-receptors (antibodies: anti-human CD184-APC (CXCR4), CD195-PE (CCR5); BD Biosciences). Cells were collected on an LSR II instrument (Becton Dickinson) and analysis was performed using FlowJo software.
9
Isolation and Staining of Mononuclear Cells
10
Phenotypic Analysis of HIV-1 gp140-Specific B Cells
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To analyze the magnitude and phenotype of the HIV-1 gp140-specific B cell responses, 2 × 106 cells from the DLNs were seeded on 96-well plates, centrifuged and incubated with Fixable Viability Stain 520 (FVS 520; BD Biosciences). After blocking Fc receptors using anti-CD16/CD32 antibody (BD Biosciences), cells were incubated with 0.3 μg/106 cells of biotinylated clade C 96ZM651 gp140 protein (Biotin-XX Microscale Protein Labeling Kit; Invitrogen) for 30 min at 4°C in the dark. After washing, cells were incubated with the following fluorochrome-conjugated antibodies for surface markers: CD3-FITC, B220-PE-Cy7, IgD-APC-H7, CD38-PerCP-Cy5.5, IgG1-BV421, GL7-Alexa647, IgM-PE-CF594, and CD19-Alexa700 (all from BD Biosciences).
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