For surface staining, cells were washed once with FACS buffer (PBS containing 0.1% BSA and 0.02% sodium azide) and cells were incubated for 15 min with the antibody mixture in FACS buffer. Cell surface staining was performed using the following antibodies: anti-CD3 (UCHT1, eBioscience™), anti-CD4 (RPA-T4, BioLegend, San Diego, USA), anti-CD8 (RPA-T8, BioLegend), anti-CD56 (HCD56, BioLegend), anti-HLA-DR (L243, BioLegend), anti-CD107a (H4A3, BioLegend), and anti-CD253/Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL) (RIK-2, BD). The Fixable Viability Dye (FVD) eFluor™ 780 (eBioscience™) was used to exclude dead cells from the analysis. Cells were washed with FACS buffer and fixated with 4% PFA for up to 2 h. Cells were washed twice with Intracellular Staining Perm Wash Buffer (BioLegend) and incubated for 20 min with anti-GzmB (GB11, BD) and anti-IFNγ (XNG1.2, BD) in Intracellular Staining Perm Wash Buffer. Cells were washed again twice with Intracellular Staining Perm Wash Buffer, collected in FACS staining buffer, and stored at 4°C until acquisition. Samples were acquired with a BD LSR II flow cytometer with a HTS module and data were analyzed using FACSDiva and FlowJo Version 10.8 (both BD Becton Dickinson, Heidelberg, Germany).
Anti cd56 hcd56
Manufactured by BioLegend
Sourced in United States
Anti-CD56 (HCD56) is a monoclonal antibody that recognizes the CD56 (NCAM1) cell surface antigen. CD56 is commonly expressed on natural killer (NK) cells, a subpopulation of T cells, and some other cell types. This antibody can be used to identify and characterize CD56-positive cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd8 rpa t8, by BioLegend (2 mentions)
Anti cd3 ucht1, by Thermo Fisher Scientific (2 mentions)
Anti cd4 rpa t4, by BioLegend (2 mentions)
Lsr 2 flow cytometer, by BD (2 mentions)
Anti cd3 okt3, by BioLegend (2 mentions)
Anti cd16 3g8, by BioLegend (2 mentions)
Fvd efluor780, by Thermo Fisher Scientific (1 mentions)
Intracellular staining perm wash buffer, by BioLegend (1 mentions)
Anti cd107a h4a3, by BioLegend (1 mentions)
Rik 2, by BD (1 mentions)
Anti hla dr l243, by BioLegend (1 mentions)
Facsdiva, by BD (1 mentions)
Anti cd4 okt4, by BioLegend (1 mentions)
Live dead fixable blue, by Thermo Fisher Scientific (1 mentions)
Anti cd56 5.1h11, by BioLegend (1 mentions)
Anti cd16 3g8, by BD (1 mentions)
Anti cd14 m5e2, by BioLegend (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Ef660, by Thermo Fisher Scientific (1 mentions)
Anti cd8 sk1, by BD (1 mentions)
7 protocols using anti cd56 hcd56
1
Comprehensive Immune Cell Profiling
2
Comprehensive Flow Cytometry Analysis of PBMCs
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PBMCs were isolated from buffy coat as described above. Freshly isolated PBMCs, frozen and thawed PBMCs, dyed PBMCs, and PBMCs exposed to 100 ng/ml LPS (Sigma, USA) were analyzed using flow cytometry. PBMCs were stained at 4 °C with the following antibodies: anti-CD56 (5.1H11, Biolegend, USA), anti-CD56 (HCD56, Biolegend, USA), anti-CD3 (OKT3, Biolegend, USA), anti-CD16 (3G8, BD Bioscience, USA), anti-CD4 (OKT4, Biolegend, USA), anti-CD8a (SK1, Biolegend, USA), anti-CD14 (M5E2, Biolegend, USA), Live Dead Fixable Blue (Thermofisher, USA). Samples were acquired using a LSRII SORP device (BD Bioscience, USA) and analyzed with Flowjo (version 10.8.1, BD, USA).
3
Phenotypic Profiling of PBMCs
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PBMCs were washed with PBS plus 2% FBS (Gibco, Grand Island, NY), and then Fc blocking reagent (Miltenyi Biotec, Inc., Auburn, CA) was added, followed by extensive wash. Cells were then incubated for 30 min on ice with anti-CD3 (OKT3) (BioLegend), anti-CD8 (SK1) (BD), anti-CD56 (HCD56) (BioLegend) and live/dead fixable aqua dye (eF660, eBioscience), washed twice with PBS plus 2% FBS and then stored at 4°C until acquired by FACS Verse (BD Biosciences, San Jose, CA). Data were analyzed using FlowJo software (Version 10.0.8, Tree Star Inc., Ashland, Or).
4
Flow Cytometry Analysis of Prostate Cells
5
Phosphoflow analysis of IFN-induced STAT signaling
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For phosphoflow analysis cells were stimulated with 2000 U/ml IFNα2, IFNα14, IFNβ, or left unstimulated (-IFN) for 15 min at 37°C. Surface staining was performed simultaneously to IFN stimulation with the following antibodies: anti-CD3 (UCHT1, eBioscience™), anti-CD4 (RPA-T4, BioLegend), anti-CD8 (RPA-T8, BioLegend), anti-CD56 (HCD56, BioLegend), as well as FVD. After stimulation cells were immediately fixated with pre-warmed Fixation Buffer (BioLegend) at 37°C. Cells were then permeabilized with pre-chilled TruePhos™ Perm Buffer (BioLegend) at -20°C for 1 h. Subsequently, cells were washed twice with Intracellular Staining Perm Wash buffer and the following antibodies were added to the cells: anti-STAT1 pTyr702 (Miltenyi Biotec); anti-STAT3 pSer727 (Miltenyi Biotec), and anti-STAT3 pTyr705 (eBioscience™), anti-STAT5 pTyr694 (BioLegend). After a 30 min incubation, cells were washed twice with FACS Intracellular Staining Perm Wash buffer and stored at 4°C until acquisition. Samples were acquired with a BD LSR II flow cytometer with a HTS module and data were analyzed using FACSDiva and FlowJo Version 10.8.
6
Isolation and Sorting of NK Cell Subsets
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PBMCs were isolated from buffy coat preparations by standard Lymphoprep (STEMCELL Biotechnology) gradient centrifugation and stored in fetal calf serum (FCS) 10% DMSO in liquid nitrogen. NK cells were pre-sorted from frozen PBMCs samples by magnetic bead isolation using the NK Cell Isolation Kit (MACS Technology, Miltenyi Biotec) or the NK Cell Enrichment Kit (EasySepTM from STEMCELL Biotechnology). Enriched NK cells were then stained with anti-CD3 (UCHT1), anti-CD7 (M-T701), anti-CD56 (HCD56), and anti-CD16 (3G8) (all from BioLegend) and CD56dim and CD56neg NK cells were sorted on a FACSAria (BD Biosciences) cell sorter. Sorted cells were cultured in complete medium (RPMI 1640 supplemented with 10% heat-inactivated FCS, 100 U/ml Penicillin, 100 mg/ml Streptomycin, and 2 mM L-glutamine; all from Invitrogen) at 37°C for 2h prior to the functional assays. Sorted NKs were used in Figure 1D, 1E, 1F ; Figure 2H, 2J ; Figure 3E ; Supplementary Figure S2E . All other experiments were performed on bulk PBMCs.
7
Cytokine Response Profiling of SARS-CoV-2 Stimulation
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PBMCs or expanded T cell lines were stimulated for 5 h (or 7 h) at 37°C with or without SARS-CoV-2 peptide pools (2 µg/ml). After 1 h (or 3 h), 10 µg/ml brefeldin A (Sigma-Aldrich) and 1× monensin (BioLegend) were added. Cells were stained with the yellow LIVE/DEAD fixable dead cell stain kit (Invitrogen) and surface marker: anti-CD3 (SK7 or OKT3; BioLegend), anti-CD4 (SK3), anti-CD8 (SK1), anti-CD14 (TUK4; Miltenyi Biotec), anti-CD16 (3G8; BioLegend), anti-CD19 (SJ25-C1), and anti-CD56 (HCD56; BioLegend). Cells were subsequently fixed and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences-PharMingen) and stained with anti–IFN-γ (25723; R&D Systems), anti–IL-2 (MQ1-17H12), anti–IL-6 (MQ2-13A5), anti–IL-10 (JES3-19F1), and anti–TNF-α (MAb11) antibodies and analyzed on a BD-LSR II FACS Scan. Data were analyzed by FlowJo (BD Biosciences). Antibodies were purchased from BD Biosciences-PharMingen unless otherwise stated.
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