Multiparametric Characterization of NK Cells

One-step staining of cell-surface antigens was performed using fluorochrome-conjugated primary antibodies as previously described (10 (link), 18 (link), 33 (link)). For the analysis of blood NK cells two antibody panels were constructed around CD56-FITC, CD16-APC, and CD3-PE-Cy7 (BD Bioscience, San Jose, CA) antibodies. NK cell activation receptors were evaluated with CD69-BV421, NKp30-BV711 (BD Bioscience), and NKp44-PerCP eFluor 710 (eBioscience, San Diego, CA) antibodies. Chemokine expression levels were tested using CXCR1-PE (R&D Systems, Minneapolis, MN), CXCR3-BV421, and CCR7-BV510 (BD Bioscience) antibodies. For the TINK analysis, Zombie NIR (BioLegend), CD45 BUV395, CD56 BV510, CD16 BUV737 (BD Bioscience), and CD3 PE-Vio770 (Miltenyi Biotec) antibodies were used. Suitable IgG controls were acquired from the same vendors. FACS analyses were performed using the BD LSRFortessa™ cell analyzer, and analyzed using FlowJo v10 (FlowJo, LLC; Ashland, OR) software.

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Vujanovic L., Chuckran C., Lin Y., Ding F., Sander C.A., Santos P.M., Lohr J., Mashadi-Hossein A., Warren S., White A., Huang A., Kirkwood J.M, & Butterfield L.H. (2019). CD56dim CD16− Natural Killer Cell Profiling in Melanoma Patients Receiving a Cancer Vaccine and Interferon-α. Frontiers in Immunology, 10, 14.

Publication 2019

CD56-FITC CD16-APC CD3-PE-Cy7 CD69-BV421 NKp30-BV711 NKp44-PerCP eFluor 710 CXCR1-PE CXCR3-BV421 CCR7-BV510 Zombie NIR CD45 BUV395 CD56 BV510 CD16 BUV737 CD3 PE-Vio770

Antibodies Antibody Blood panels Cell Cell surface antigens Chemokine Cxcr3 Fitc Fluorochrome Nk cell receptors Nk cells Nkp44

Corresponding Organization : University of Pittsburgh

Other organizations : Nanostring Technologies (United States)

Top 5 similar protocols

Variable analysis

independent variables

Use of fluorochrome-conjugated primary antibodies
Antibody panels constructed around CD56-FITC, CD16-APC, and CD3-PE-Cy7 (BD Bioscience, San Jose, CA) antibodies
Evaluation of NK cell activation receptors with CD69-BV421, NKp30-BV711 (BD Bioscience), and NKp44-PerCP eFluor 710 (eBioscience, San Diego, CA) antibodies
Testing of chemokine expression levels using CXCR1-PE (R&D Systems, Minneapolis, MN), CXCR3-BV421, and CCR7-BV510 (BD Bioscience) antibodies
Use of Zombie NIR (BioLegend), CD45 BUV395, CD56 BV510, CD16 BUV737 (BD Bioscience), and CD3 PE-Vio770 (Miltenyi Biotec) antibodies for TINK analysis

dependent variables

Cell-surface antigen expression levels
NK cell activation receptor expression
Chemokine expression levels

control variables

Suitable IgG controls acquired from the same vendors

Annotations

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