PBMCs were stimulated overnight with 1 ng/mL interleukin (IL) 15 (R&D). These were then incubated with K562 cells at an Effector:Target (E:T) ratio of 10:1 in the presence of CD107a-AF647 antibody (eBioscience), with Golgi Stop (BD Biosciences) and stained with CD3-Pacific Blue and CD56-PECy7 or with anti-interferon (IFN)γ-FITC and antitumour necrosis factor (anti-TNF)α-PE (BioLegend).
Cd56 pe cy7
Manufactured by BioLegend
Sourced in United States
CD56-PE-Cy7 is a fluorescently-labeled antibody that binds to the CD56 surface antigen. CD56 is expressed on natural killer cells, a subset of T cells, and some other cell types. This antibody-fluorophore conjugate can be used to identify and quantify CD56-positive cells in flow cytometry applications.
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Lab products found in correlation
Cd3 percp, by BioLegend (5 mentions)
Golgistop, by BD (5 mentions)
Facscanto 2, by BD (3 mentions)
Il 15, by R&D Systems (3 mentions)
Rea147 fitc, by Miltenyi Biotec (3 mentions)
Ch l pe, by BD (3 mentions)
Facsaria, by BD (3 mentions)
Cd25 pe, by BioLegend (2 mentions)
Cd8 pe cy7, by BioLegend (2 mentions)
Cd14 pe cy7, by BioLegend (2 mentions)
Cd3 pe cy7, by BioLegend (2 mentions)
7 aad, by BioLegend (2 mentions)
Cd45 pacific blue, by BioLegend (2 mentions)
Cd49a pe, by BD (2 mentions)
Cd3 bv510, by BioLegend (2 mentions)
Cxcr6 percp cy5, by BioLegend (2 mentions)
Il 12, by Thermo Fisher Scientific (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Cd62p fitc, by BD (2 mentions)
Cd41a pecy5, by BD (2 mentions)
30 protocols using cd56 pe cy7
1
Cytotoxic NK Cell Activation Assay
2
Treg and NK-cell Immunophenotyping
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PBMCs were thawed and stained with either Treg or NK-cell discriminating antibodies. The Treg panel was comprised of the following extracellular antibodies: CD3-AF700, CD4-eFluor780, CD8-PE-Cy7, CD25-PE, CD127-BV421, CD45RO-BV711 (Biolegend, Biolegends, Koblenz; Germany). For intracellular staining, cells were permeabilized after extracellular staining, using the eBioscience kit (Thermo Fisher Scientific, Darmstadt, Germany) and stained for FOXP3 expression. The NK-cell panel comprised of CD3-AF700, CD19-eFluor780, CD56-PE-Cy7, CD16-BV510, CD45RO-BV711, TCRVα24-PE, TCRVβ11-FITC (Biolegend). All samples were measured within 24 h after staining on a BD LSR Fortessa (BD Biosciences, Heidelberg, Germany). All flow cytometry data were analyzed with FlowJo software version 10.6.0 (Tree Star, Ashland, OR, USA). Output CSV documents were further analyzed using RStudio (version 1.2.1335).
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
NK Cell Cytotoxicity Assay
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1 × 105 NK cells were added to tumor cell lines at an E/T ratio of 1 : 1 in 200 μl, spun down at 300 r.p.m. for 2 min and incubated in the presence of FITC-conjugated anti-CD107a antibody or isotype control antibody (BioLegend). After 30 min, 1 μg of GolgiStop (BD Biosciences) was added for an additional period of 3.5 h. As a positive control for NK cell degranulation, NK cells were incubated with 50 ng/ml PMA and 1 mM ionomycin (Sigma-Aldrich). When indicated, NK cells were incubated with 10 μg/ml of anti-NKG2D (1D11) or isotype control for 20 min before the addition to target cells. Cell suspensions were washed, stained for CD45-Pacific blue, CD3-PE and CD56-PE Cy7 (all from BioLegend) on ice in FACS buffer for 30 min, washed and briefly incubated with a 1 : 40 dilution of 7-AAD (BioLegend). Cells were measured on a FACS Canto II (BD Biosciences) and analyzed using FlowJo 9.3.2 software (Treestar).
5
Cytokine-Stimulated Immune Cell Profiling
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PBMCs and liver MNCs were stimulated for 12 h with IL‐12 10 ng/ml (PeproTech) or IL‐15 1 ng/ml (R&D Systems) to examine IFNγ and TNFα production respectively. An unstimulated control was included. BD GolgiStop™ (BD Biosciences) was added (4 µl/6 ml culture medium) for the last 4 h. Surface staining was performed for CD3‐BV510 (Biolegend®), CD56‐PE‐Cy7 (Biolegend®), CXCR6‐PerCP/Cy5.5 (Biolegend®), and CD49a‐PE (BD Biosciences). Cells were fixed and permeabilised (BD Cytofix/Cytoperm™ Plus Kit, BD Biosciences) prior to incubation with IFNγ (B27, APC, Biolegend®) or TNFα (MAb11, FITC, Biolegend®).
6
Flow Cytometric Analysis of NK Cell Receptors
7
Platelet Activation Assay Protocol
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Paraformaldehyde was obtained from Affymetrix (Santa Clara, CA). Anti-GITR, anti-GITRL antibodies and the respective isotype control were from R&D Systems (Minneapolis, MN). CD19-FITC, CD41a-PECy5, CD41a-PE, PAC-1-FITC, CD61-FITC and CD62P-FITC were from BD Pharmingen (San Diego, CA), CD3-APC/Fire and CD56-PeCy7 were from Biolegend (San Diego, CA). The goat anti-mouse PE conjugate was from Dako (Glostrup, Denmark). Biocoll Separating Solution was purchased from Biochrom AG (Berlin, Germany). VPA was from Sigma-Aldrich (St. Louis, MO). Thrombin Receptor Activator Peptide 6 (TRAP-6), collagen and ADP were purchased from Sigma-Aldrich (St. Louis, MO).
8
Comprehensive Immunophenotyping of Tumor and T Cells
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Tumor cells and T cells were phenotyping with CD3‐APC (OKT3, UCH1), CD19‐APC CY7 (SJ26C1), CD4‐PE Cy7 (RPA‐T4, A161A1), CD8‐pacific blue (RPA‐T8, SK1), CD10‐APC (HI10a), CD22‐PE (HIB22), CD38‐PECy7 (HTT2), CD56‐PE CY7 (MEM‐188), CD62L‐BV510 (DREG‐56), PDL1‐PE (29E.2A3), PD1‐FITC (EH12.2H7), streptavidin‐APC‐Cy7, and 7AAD, which were purchased from BioLegend. CD20‐FITC (2H7), CD45RA‐FITC (HI100), and CD62L‐PE (DREG‐56) were purchased from BD. CD137‐PE, CD34‐APC, and antibiotin‐PE were purchased from MACS. The percentage of CAR‐CD19 was determined by biotinylated Erbitux. All cells were analyzed MACSquant with a filter set for eight fluorescence signals and analyzed with FlowJo software (Tree Star).
9
Immunophenotyping of Breast Cancer Co-Cultures
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Cocultures were isolated 3 days after establishment. After pooling 6 co-cultures, breast cancer spheroids were extensively washed and trypsinized to obtain a cell suspension. Fc receptors were blocked with Human TruStain FcX™ (Fc Receptor Blocking Solution, cat num. 422301, BioLegend) for 10 min at RT, prior to incubation with the following antibodies: CD3-APC-Cy7, CD4-APC, CD14-BV510, CD15-BV650, CD16-PerCP-Cy5.5, CD56-Pe-Cy7 and CD86-BV786 (all from BioLegend) for 30 min at 4°C in the dark. Cells were washed and fixed in 1% formaldehyde prior to analyzing in the flow cytometer (FACS LSRII, BD Biosciences). Immunophenotyping of different cell subsets were defined based on described expression combinations gated on the live singlet lymphocytes. Data was analyzed using the FlowJo software (BD Biosciences).
10
Single-cell analysis of heX-embryoid
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To prepare a single-cell solution of heX-embryoid, the culture was treated for 45 min with Collagenase C solution (3 mg ml−1 StemCell Technologies) followed by 15 min of treatment with Accuatse (Sigma). FC block solution (Thermo Fischer) was added to the samples followed by 10 min of incubation on ice. Next, the antibody mix (final dilution of 1:400) was added to the samples followed by 30 min incubation on ice. Cells were analysed using an LSR II flow cytometer (BD Bioscience) using 7-AAD (BD Pharmingen) for dead cell staining. The antibodies used were CD34-APC (Clone 581, Biolegend), KIT-BV421 (Clone 104D2, BD Bioscience), CD43-PE (Clone CD43-10G7, Biolegend), CD235ab-PE-Cy7 (Clone HIR2, Biolegend), CD33-BV605 (Clone P67.6, Biolegend), CD42b-AF700 (Clone HIP1, Biolegend), CD7-PE-Cy7 (Clone 4H9/CD7, Biolegend), CD45-Pacific Blue (Clone HI30, Biolegend), CD45-APC-Cy7 (Clone HI30, Biolegend), CD45-APC (Clone HI30, Biolegend), CD31-PE-Cy7 (Clone WM59, Biolegend), CD15-PE (Clone HI98, Biolegend), CD56-PE-Cy7 (Clone MEM-188, Biolegend) and CD49d(VLA-4)-BV605 (Clone 9F10, Biolegend). Back-gating, controls and analysis steps can be seen in Supplementary Information 1 and 2 .
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