A blood sample from immunized macaque 25328 at week 146 was collected, PBMCs were frozen in liquid nitrogen and thawed for later analyses. PBMCs were washed with 1xPBS containing 1% BSA and stained with biotinylated cyclic V3MN peptide followed by streptavidin-APC and fluorescence-conjugated Abs against cell markers CD3-PE-CF594, CD20-PE-Cy7, CD19-APC-Cy7, IgG-FITC, and IgM-BV450 (Becton Dickinson). V3-specific single positive B cells were isolated in 96-well plates by FACS gating strategy as previously described (2 (link)) (Fig. 2 ). mRNA was isolated from single B cells and reverse transcribed in the 96-well plate by addition of random hexamer primers (Invitrogen), 0.35 mM dNTP mix (Invitrogen), 10 mM DTT, 2U RNAsin (Promega), 4U Superase-In (Ambion), 0.5% v/v Igepal CA-630 (Sigma-Aldrich) and 50 U superscript III reverse transcriptase (Invitrogen). The immunoglobulin (Ig) variable genes were amplified by nested PCR and cloned into plasmids containing a constant region as described (14 (link), 15 (link)). Macrogen, Inc. sequenced the VH, Vκ, and Vλ chains. Recombinant Abs were produced by co-transfection of H/κ or H/λ plasmids into 293T Cells (2 (link)), and IgG production in culture supernatants was quantitated. Supernatants with recombinant Abs were screened for binding activity to V3MN, gp120MN, and gp41MN and mAbs specific to V3 and gp120, but not to gp41 were identified.
Cd20 pe cy7
Manufactured by BD
The CD20-PE-Cy7 is a fluorochrome-conjugated antibody that binds to the CD20 antigen expressed on the surface of B cells. It is used for the identification and enumeration of B cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 percp cy5, by BD (2 mentions)
Cd11c pe cy7, by BD (2 mentions)
Anti rabbit igg apc secondary antibody, by BD (2 mentions)
Cd123 percp cy5, by BD (2 mentions)
Accuri c6, by BD (2 mentions)
Cytofix cytoperm, by BD (2 mentions)
Cd8 pe cy7, by BD (2 mentions)
Facsaria 2, by BD (2 mentions)
Random hexamer primer, by Thermo Fisher Scientific (1 mentions)
Superase in, by Thermo Fisher Scientific (1 mentions)
Dntp mix, by Thermo Fisher Scientific (1 mentions)
Igepal ca 630, by Merck Group (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rnasin, by Promega (1 mentions)
Hla dr pb, by BioLegend (1 mentions)
Live dead fixable aqua stain, by Thermo Fisher Scientific (1 mentions)
Cd11c apc, by BD (1 mentions)
Cd56 pe, by BD (1 mentions)
Cd3 apc cy7, by BD (1 mentions)
Cd14 percp cy5, by BD (1 mentions)
10 protocols using cd20 pe cy7
1
Isolation and Characterization of V3-specific B Cells
2
Investigating PBMC Infection by Recombinant MVA
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PBMC were isolated from blood obtained from healthy humans and seeded into 96-wells low-adherent v-bottom plates at 50.000 cells/well. Subsequently, cells were inoculated with rMVA-GFP at incrementing MOI (range MOI 0.01–100, 3-fold titration), for 1 h, washed and incubated for 24 h in Roswell Park Memorial Institute (RPMI) 1640 medium (Lonza) supplemented with 10% heat-inactivated foetal bovine serum (HI-FBS, Greiner Bio-One), 100 μg/ml penicillin, 100U/ml streptomycin (Lonza) and 2mM L-Glutamine (Lonza) (P/S/G) at 37 °C. After 24 h, cells were harvested and stained with CD3APC/Cy7 (BD Pharmingen), CD11cAPC (BD Pharmingen), CD14PerCP/Cy5.5 (BD Pharmingen), CD56PE (BD Pharmingen), CD20PE/Cy7 (Becton Dickinson) and HLA-DRPB (Biolegend), in combination with LIVE/DEAD aqua fixable stain (Invitrogen). Cells were analysed on a flow cytometer (FACS Canto II) and the percentage of infected cells (GFP+) within the respective subsets was determined using FACS Diva software (BD Biosciences).
3
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.
4
Antigen-specific B Cell Sorting
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Cryopreserved 107 PBMC were thawed into 1 ml preheated RPMI1640, centrifuged at 300 × g for 5 min, resuspended in 500 μl FACS buffer (PBS + 2% FBS), and incubated with 200 nM his-tagged antigen (gH/gL) for 45 min at 4 °C. The PBMC were then washed two times with 1 ml FACS buffer and resuspended in 100 μl FACS buffer. The PBMC were stained with the following antibodies: CD3-PE-Cy5 (BD Biosciences Cat#555341) at a 1:25 dilution, CD14-PE-Cy5 (eBioscience Cat#15-0149-42) at a 1:50 dilution, CD16-PE-Cy5 (BD Biosciences Cat#555408) at a 1:25 dilution, CD235a-PE-Cy5 (BD Biosciences Cat#559944) at a 1:100 dilution, CD19-APC-Cy7 (BD Biosciences Cat#348794) at a 1:100 dilution, CD20-PE-Cy7 (BD Biosciences Cat#335793) at a 1:200 dilution, IgG-FITC (BD Biosciences Cat#555786) at a 1:25 dilution, and anti-his-PE (BioLegend Cat#362603) at a 1:20 dilution for 30 min at 4 °C. The PBMC were washed three times with 1 ml FACS buffer and resuspended in 500 μl FACS buffer, then subjected to FACS on a BD FACS Aria II (BD Biosciences).
Antigen-positive B cells (CD3-, CD14-, CD16-, CD235a-, CD19+, CD20+, IgG+, PE+) were sorted individually into 96-well PCR vital-plates containing 20 μl first strand buffer (5 μl first strand buffer, 0.5 μl of RNase inhibitor (Invitrogen Cat#10777019), 1.25 μl of 100 μM DTT, 0.06 μl of IGEPAL (Sigma Cat#I8896).
Antigen-positive B cells (CD3-, CD14-, CD16-, CD235a-, CD19+, CD20+, IgG+, PE+) were sorted individually into 96-well PCR vital-plates containing 20 μl first strand buffer (5 μl first strand buffer, 0.5 μl of RNase inhibitor (Invitrogen Cat#10777019), 1.25 μl of 100 μM DTT, 0.06 μl of IGEPAL (Sigma Cat#I8896).
5
SARS-CoV-2 Detection in Tonsil TMNCs
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Frozen purified TMNCs were analyzed by flow cytometry after a 30-min staining at 4°C using antibodies for CD4 (PerCP-Cy5.5), CD8 (PE-Cy7), CD11c (PE-Cy7), CD14 (PerCP), CD20 (PE-Cy7), and CD123 (PerCP-Cy5.5) (BD Pharmingen). Cells were then washed, permeabilized, and fixed with BD Cytofix/Cytoperm. Intracellular SARS-CoV-2 was stained with rabbit anti-SARS-CoV-2 NP antibody, followed by anti-rabbit IgG-APC secondary antibody (BD Pharmingen). TMNCs from tonsils RT-qPCR negative for SARS-CoV-2 were used as negative controls. Cell preparations stained only with the IgG-APC secondary antibody were used for calibration of PE acquisition. Acquisitions were performed in fixed cells in a flow cytometer (BD Accuri C6; BD Biosciences) and then analyzed using FlowJo software (Tree Star).
6
Multiparameter Flow Cytometry for B-ALL MRD Detection
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MPFC was performed with a panel of antibodies designed for B‐ALL. The diagnostic panel for B‐ALL contained CD45, CD7, CD19, CD13, CD33, CD34, CD117, HLA‐DR, CD10, cMPO, cCD3 and cCD79a and its extended panel included CD66c, CD22, CD20, CD58, CD38, CD123, CD45, cytoplasmic heavy chain of immunoglobulin M(cu) and cytoplasmic TDT (BD Bioscience; San Jose, CA; Beckman‐Counter; Indianapolis, IN).
15 (link) FACS Aria II (BD Biosciences) with Diva program was used to acquire and analyze the data. MRD panel included CD58‐FITC, KORSA‐PE, CD34‐Percp, CD20‐PE‐cy7, CD10‐APC, CD19‐APC‐H7, CD38‐V450 and CD45‐V500 (BD Bioscience and Beckman‐Counter).
Negative MRD by MPFC was defined as <10−4 blasts (0.01%) in bone marrow samples and Flow‐MRD positivity was defined as >10−4 blasts (0.01%) in bone marrow. In our study, patients with sustained undetectable MRD were defined according to negative MRD results observed at the end of consolidation and any of the previous time points.
15 (link) FACS Aria II (BD Biosciences) with Diva program was used to acquire and analyze the data. MRD panel included CD58‐FITC, KORSA‐PE, CD34‐Percp, CD20‐PE‐cy7, CD10‐APC, CD19‐APC‐H7, CD38‐V450 and CD45‐V500 (BD Bioscience and Beckman‐Counter).
Negative MRD by MPFC was defined as <10−4 blasts (0.01%) in bone marrow samples and Flow‐MRD positivity was defined as >10−4 blasts (0.01%) in bone marrow. In our study, patients with sustained undetectable MRD were defined according to negative MRD results observed at the end of consolidation and any of the previous time points.
7
SARS-CoV-2 Detection in Tonsil TMNCs
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Frozen purified TMNCs were analyzed by flow cytometry after a 30-min staining at 4°C using antibodies for CD4 (PerCP-Cy5.5), CD8 (PE-Cy7), CD11c (PE-Cy7), CD14 (PerCP), CD20 (PE-Cy7), and CD123 (PerCP-Cy5.5) (BD Pharmingen). Cells were then washed, permeabilized, and fixed with BD Cytofix/Cytoperm. Intracellular SARS-CoV-2 was stained with rabbit anti-SARS-CoV-2 NP antibody, followed by anti-rabbit IgG-APC secondary antibody (BD Pharmingen). TMNCs from tonsils RT-qPCR negative for SARS-CoV-2 were used as negative controls. Cell preparations stained only with the IgG-APC secondary antibody were used for calibration of PE acquisition. Acquisitions were performed in fixed cells in a flow cytometer (BD Accuri C6; BD Biosciences) and then analyzed using FlowJo software (Tree Star).
8
Quantification of Tetramer-Specific CD8+ T Cells
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PBMC and tissue-derived MNC were thawed and subjected to Live/Dead Aqua fluorescent reactive dye (ThermoFisher) in PBS for 30 mint at RT. The cells were washed cells with RPMI + 10% FBS and subsequently incubated with 3µg/mL A01-CM9 tetramers (NIH Tetramer Core Facility and MBL International) in PBS + 2% FBS at room temperature for 35 min at 2 × 106 cells/100 µL in polypropylene FACS tubes. The cells were then incubated at 4 °C with antibodies directed to cell surface markers for additional 30 min. After 2 washes with PBS + 2% FBS, the cells were fixed with 200 µL of stabilizing fixative (BD Biosciences) at room temperature. Tetramer + CD8 + T cells were quantified with the aid of a Fortessa flow cytometer (BD) by gating on live, singlets, CD45 + , CD8 + CD3 + CD14- CD20- CD4- cells present in the lymphocyte gate (CD45-PerCP, BD Biosciences, catalog number 558411, 1:10 dilution; CD8-BUV395, BD Biosciences, catalog number 563795, 1:50 dilution; CD3-FITC BD Biosciences, catalog number 556611, 1:5 dilution; CD14-PE-Cy7, BD Biosciences, catalog number 557742, 1:100 dilution; CD20-PE-Cy7, BD Biosciences, catalog number 560735, 1:50 dilution; CD4-BV785, Biolegend, catalog number 317442, 1:100 dilution).
9
Multiparametric Flow Cytometry of Immune Cells
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Whole blood or PBMC were stained with antibody mix comprising anti-human CD3-Bv421, CD4-PE, CD8-APC, CD14-APCH7, CD19-Bv480, CD20-PECy7, and CD45-FITC (clones UCHT1, SK3, SK1, MphiP9, SJ25C1, L27, 2D1, respectively) and 7-AAD (all from BD Biosciences). For whole blood, BD Trucount™ tubes were used according to the manufacturer’s instructions (Figure S1
10
Isolation and Phenotyping of Plasma Cells
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At 3 days post-infection cells were collected and pelleted at 1400 rpm for 3 minutes into 12x75mm round bottom tubes. Cells were resuspended in 200μl PBS containing (0.4ng/ml) fixable viability stain (BD 565388) and incubated on ice for 15 minutes. Cells were pelleted and resuspended in 200μl cold MACS buffer containing 5% FBS (Sort Block) and incubated on ice for 10 minutes after which 200μl cold MACS buffer was added. Cells were pelleted and resuspended in MACS buffer containing B cell phenotype panel as follows for 15 minutes on ice: (volumes indicated are for each 1(10)^6 cells and were scaled depending upon the number of cells being stained for sorting). For single cell sorting of plasma cells the panel was follows: CD19-PerCPCy5.5 (5μl, BD 561295), CD20-PE-Cy7 (5ul, BD 560735), and CD138-APC (5μl, Biolegend 352307). For other lineages the panel was as follows: CD19-PerCPCy5.5 (5μl, BD 561295), CD38-APC (20μl, BD 555462), IgD-PE-Cy7 (5, BD 561314), CD27-PE-Cy5 (5μl eBioscience 15-0279-42). After incubation, 500μl MACS buffer was added and pelleted lymphocytes were washed with a further 500μl of MACS buffer prior to being resuspended in 200μl MACS buffer and put through a cell strainer before sorting using the 70-micron nozzle on a BD FACSAria Fusion Cell Sorter.
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