Antibodies used for flow cytometry analysis included: CD2-Tri-Color (TC); CD4-Phycoerythrin (PE) and CD4Allophycocyanin (APC); CD28-Fluorescein Isothiocyanate (FITC); CD8-TC; CD19-APC; CD45RA-APC; CD16-FITC from Life Technologies (Grand Island, NY); CD14-PE; CD27-PE; and IgM-FITC from BD Biosciences (San Jose, CA). Freshly thawed PBMCs from each visit were stained with three to five antibodies: T cells (CD2, CD4, CD8, CD45RA, and CD28); B cells (CD19, IgM, and CD27); NK cells (CD16). The data were collected on a BD FACSCalibur or BD FACSCanto II, and analyzed by Cell-Quest (BD Biosciences) and FlowJo. The gating strategies were presented in Additional file 1 : Figure S1.
Cd27 pe
Manufactured by BD
Sourced in United States, United Kingdom
CD27-PE is a fluorescently-labeled antibody that binds to the CD27 cell surface receptor. CD27 is a member of the tumor necrosis factor receptor superfamily and is expressed on various immune cells, including T cells and B cells. The PE (Phycoerythrin) fluorescent label allows for the detection and analysis of CD27-expressing cells using flow cytometry or other fluorescence-based techniques.
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Lab products found in correlation
Facscalibur, by BD (5 mentions)
Cd38 fitc, by BD (4 mentions)
Facscanto 2, by BD (3 mentions)
Cellquest, by BD (3 mentions)
Igm fitc, by BD (3 mentions)
Facsaria fusion, by BD (3 mentions)
Facsdiva software, by BD (3 mentions)
Cd4 percp, by BD (3 mentions)
Cd8 fitc, by BD (3 mentions)
Cd3 percp, by BD (3 mentions)
Igd fitc, by BD (3 mentions)
Cd38 pe cy7, by BD (3 mentions)
Cd19 apc, by Thermo Fisher Scientific (2 mentions)
Cd14 pe, by BD (2 mentions)
Cd45ra apc, by Thermo Fisher Scientific (2 mentions)
Cd8 tc, by Thermo Fisher Scientific (2 mentions)
Cd8 apc, by BD (2 mentions)
Cd3 pe cy7, by BioLegend (2 mentions)
Cd25 pe, by BD (2 mentions)
Cd123 pe, by BD (2 mentions)
41 protocols using cd27 pe
1
Comprehensive Immune Cell Phenotyping
2
Isolation and Characterization of Naive T Cells
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After thawing, PBMCs were stained with CCR7 BV650 (Clone 3D12; BD Biosciences), CD95 FITC (Clone DX2; BD Biosciences), CD27 PE (Clone M-T271; BD Biosciences), CD8 APC (Clone RPA-T8; BD Biosciences), CD4 APC-Cy7 (Clone RPA-T4; BD Biosciences), CD45RA ECD (Clone IM271111; Beckman Coulter), CD57 PB (Clone HCD57; Biolegend), CD49d PeCY7 (Clone 9F10; Biolegend), and eFluor 506 for viability (Biolegend). Naive CD4+ and CD8+ T cells, defined as CD45RA+, CD27+ CCR7+, CD95−, CD49d−, were sorted on an ARIAIII (Becton Dickinson). DNA and RNA from sorted cells were extracted using AllPrep DNA/RNA Micro kit (Qiagen) according to manufacturer instruction.
3
Isolation and Expansion of SARS-CoV-2-Specific B Cells
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Blood samples were screened for SARS-CoV-2 RNA and for antibodies against HIV, HBV and HCV. Peripheral blood mononuclear cells (PBMCs) were isolated from heparin-treated whole blood by density gradient centrifugation (Ficoll-Paque PREMIUM, Sigma-Aldrich). After separation, PBMC were stained with Live/Dead Fixable Aqua (Invitrogen; Thermo Scientific) in 100 μL final volume diluted 1:500 at room temperature RT. After 20 min incubation cells were washed with PBS and unspecific bindings were saturated with 50 μL of 20% normal rabbit serum (Life technologies) in PBS . Following 20 min incubation at 4°C cells were washed with PBS and stained with SARS-CoV-2 S-protein labeled with Strep-Tactin®XT DY-488 (iba-lifesciences cat# 2-1562-050) for 30 min at 4°C. After incubation the following staining mix was used CD19 V421 (BD cat# 562440), IgM PerCP-Cy5.5 (BD cat# 561285), CD27 PE (BD cat# 340425), IgD-A700 (BD cat# 561302), CD3 PE-Cy7 (BioLegend cat# 300420), CD14 PE-Cy7 (BioLegend cat# 301814), CD56 PE-Cy7 (BioLegend cat# 318318) and cells were incubated at 4°C for additional 30 min. Stained MBCs were single cell-sorted with a BD FACS Aria III (BD Biosciences) into 384-well plates containing 3T3-CD40L feeder cells and were incubated with IL-2 and IL-21 for 14 days as previously described (Huang et al., 2013 (link)).
4
Isolation and Sequencing of IgG+ Memory B Cells
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B cells were isolated from PBMCs using CD19 microbeads (Miltenyi Biotec) and stained with DAPI (Thermo Fisher), CD20-AF 700, IgG-APC, IgD-Pe-Cy7, IgM-FITC, and CD27-PE (all BD Biosciences) for 30 min on ice. 200,000 CD20+IgG+IgM-IgD-CD27- B cells were sorted into FBS (Sigma-Aldrich) using a BD FACSAria Fusion, and RNA of sorted B cells was isolated with the RNeasy Micro Kit (QIAGEN). cDNA was generated by template-switch reverse transcription according to the SMARTer RACE 5′/3′ manual using the SMARTScribe Reverse Transcriptase (Takara) with a template-switch oligo including an 18-nucleotide unique molecular identifier. Heavy-chain variable regions were amplified with an IgG-specific nested PCR and amplicons were used for library preparation and MiSeq 2x300 bp sequencing (Illumina). Raw NGS reads were pre-processed and assembled to final sequences as previously described (Ehrhardt et al., 2019 (link)).
5
Immunophenotypic Analysis of HCL Cells
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Multiparameter flow cytometric immunophenotype was performed as described previously [18 (link)], with sIg expression analysed on CD19+CD11cHI or CD19+CD11cHICD103+ HCL cells (Table 1 ). Additional markers were assessed using CD25-PE (BD Biosciences, Oxford, UK), CD27-PE (BD) and CD123-PE (BD) antibodies on CD19+CD11cHICD103+ cells (Table 1 ). Data were analysed using FACSDiva (BD) and FlowJo (Tree Star Inc., Ashland, OR, USA). ANXA1 detection was performed as previously described [19 (link)]. Briefly, ANXA1 was detected either by western blot or immunocytochemistry using the antibody ‘Purified mouse anti-Annexin I’, clone 29/Annexin I (BD Transduction Laboratories, Oxford, UK) (Table 1 ).
6
Flow Cytometric Analysis of Lymphocyte Phenotypes
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Cryopreserved PBMCs collected at T0 and T12 were thawed and stained (1x106 cells) with fluorochrome-labelled antibodies for the flow cytometric study of lymphocyte surface phenotypes. To check cell viability, cells were stained with 7-aminoactynomycin D (7-AAD, BD Biosciences, San Jose, California, USA) for 30 min in the dark at 4°C. Only samples with cellular viability greater than 70% were used for experiments.
The following antibodies were used: HLA-DR-FITC, CD38-PE, CCR7-PeCy7, CD45RA-PeCy5, CD27-PE, CD95-APC, α4β7integrin-APC CCR6-PeCy7, CD161-APC (BD Biosciences, San Jose, California, USA), CCR9-FITC (R&D Systems, Minneapolis, MN, USA).
We evaluated CD4+ and CD8+ activation (HLA-DR+CD38+), maturation (naïve: CCR7+CD45RA+; central memory: CCR7+CD45RA-; effector memory: CCR7-CD45RA-; terminally differentiated: CCR7-CD45RA+) and stem cell-like memory T cells (Tscm; CCR7+CD45RA+CD27+CD95+). CD4+ T-cell populations involved in mucosal immunity (CCR9+α4β7+; CCR6+CD161+) were also studied.
Cells were run on a FACS VERSE cytometer (BD Biosciences, San Jose, California, USA).
The following antibodies were used: HLA-DR-FITC, CD38-PE, CCR7-PeCy7, CD45RA-PeCy5, CD27-PE, CD95-APC, α4β7integrin-APC CCR6-PeCy7, CD161-APC (BD Biosciences, San Jose, California, USA), CCR9-FITC (R&D Systems, Minneapolis, MN, USA).
We evaluated CD4+ and CD8+ activation (HLA-DR+CD38+), maturation (naïve: CCR7+CD45RA+; central memory: CCR7+CD45RA-; effector memory: CCR7-CD45RA-; terminally differentiated: CCR7-CD45RA+) and stem cell-like memory T cells (Tscm; CCR7+CD45RA+CD27+CD95+). CD4+ T-cell populations involved in mucosal immunity (CCR9+α4β7+; CCR6+CD161+) were also studied.
Cells were run on a FACS VERSE cytometer (BD Biosciences, San Jose, California, USA).
7
Cryopreserved PBMC Characterization and Analysis
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Cryopreserved PBMC were thawed and allowed to rest overnight (37°C, 5% CO2) as previously described [41 (link), 42 (link)]. Plating of cells (1x106), staining for viability, bacteria binding (50:1—bacteria:cells ratio [43 (link)]), blocking (human IgG -25 μl of a 1 mg/ml solution; mouse IgG -25 μl of a 200 μg/ml solution) and staining of surface targets with monoclonal antibodies were performed as described in detail in [40 (link)]. Monoclonal antibodies (mAbs) against the following molecules were used: CD19-ECD (clone J3-119; Beckman Coulter -BC-), CD38-PE-Cy5 (clone LS1298-4-3; BC), CD14-QDot 655 (clone TuK; Invitrogen), CD21-BV711 (clone B-ly4; Becton-Dickinson -BD-), integrin α4β7-Alexa647 (clone ACT-1; Millennium, The Takeda Oncology Co), CD3-Alexa Fluor 700 (clone UCHT1; BD), IgD-FITC (polyclonal goat anti-sera; Southern Biotech), CD27-PE (clone L128; BD), CD40-PE-Cy7 (clone 5C3; BD), IgA-Biotin (polyclonal goat anti-sera; Southern Biotech) and Pacific Orange-Streptavidin (Invitrogen, USA). Finally, stained cells were fixed with 1% PFA in PBS until data collection in a LSRII (BD) instrument.
8
Multiparametric Flow Cytometry of PBMCs
9
Multimer-based peanut allergy analysis
10
T Cell Surface Marker Staining
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T cells were washed with PBS containing 2% FBS prior to the addition of antibodies. Cells were stained with fluorophore-conjugated antibodies specific for mouse CD3-AF700 (BD Biosciences, San Jose, CA, USA), CD8-APC (eBioscience, Waltham, MA), CD27-PE (BD Biosciences, San Jose, CA, USA), and CD44-BV421 (BD Biosciences, San Jose, CA, USA) in PBS with 2% FBS for 15 min at 4 °C. Cells were then fixed in 4% paraformaldehyde for 10 min at 4 °C and washed twice in PBS with 2% FBS. Flow cytometry was performed on a Canto II flow cytometry system (BD Biosciences, San Jose, CA) 32 (link).
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