Immunophenotyping of human NK cells

Immunophenotyping of human NK cells was performed using flow cytometry analyses. Cryopreserved peripheral blood mononuclear cells (PBMC) collected from 15 healthy normal adult donors were thawed and incubated overnight (18 h) in RPMI 1640 medium supplemented with 10% FBS, or in RPMI 1640 medium supplemented with 10% FBS and 10 ng/mL recombinant human IL-15 (Miltenyi Biotec, GmbH). The cells were then washed with PBS and stained with a viability marker (Fixable Aqua Dead Cell Stain Kit, Thermo Fisher Scientific, San Diego, CA) prior to surface and intracellular staining with fluorescently conjugated Abs using standard techniques. The staining panel used to identify NK cell subsets and phenotypes was based on the Optimized Multicolor Immunofluorescence Panel (OMIP) described previously [OMIP-007, (33 (link))]. Fluorescently conjugated Abs used for surface staining were: PE-TR-CD3, clone S4.1, Thermo Fisher Scientific; APC-H7-CD4, clone SK3, BD Biosciences, San Jose, CA; PE-Cy5-CD14, clone Tuk4, Thermo Fisher Scientific; PE-Cy5-CD19, clone SJ25-C1, Thermo Fisher Scientific; PacificBlue-CD16, clone 3G8, BD Biosciences; PE-Cy7-CD56, clone NCAM16.2, BD Biosciences; BV606-CD62L, clone DREG-56, Biolegend, San Diego, CA; FITC-HLA-DR, clone G46-6, BD Biosciences; APC-CD57, clone HCD57, Biolegend; and BV785-CD69, clone FN50, Biolegend. Intracellular staining was performed with BV711-Perforin, clone dG9, Biolegend, and PE-Granzyme B, clone GB11, BD Biosciences. Quantum™ Simply Cellular^® beads (Bangs Laboratories, Inc., Fishers, Indiana) were used to determine the Ab binding capacity (ABC) of perforin and granzyme within cells according to the manufacturer's recommended procedure. Data analysis was performed using FlowJo software (v10.5.3).

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Fisher L., Zinter M., Stanfield-Oakley S., Carpp L.N., Edwards R.W., Denny T., Moodie Z., Laher F., Bekker L.G., McElrath M.J., Gilbert P.B., Corey L., Tomaras G., Pollara J, & Ferrari G. (2019). Vaccine-Induced Antibodies Mediate Higher Antibody-Dependent Cellular Cytotoxicity After Interleukin-15 Pretreatment of Natural Killer Effector Cells. Frontiers in Immunology, 10, 2741.

Publication 2019

Adult Cd62l Cells Clone Donors Fitc Flow cytometry Granzyme Granzyme b Hla dr Human Il 15 Immunofluorescence Intracellular Nk cell Perforin Peripheral blood mononuclear cells Phenotypes Stain

Corresponding Organization : Duke University Hospital

Other organizations : Perinatal HIV Research Unit, University of the Witwatersrand, Desmond Tutu HIV Foundation, University of Cape Town, University of Washington

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Variable analysis

independent variables

Incubation of cryopreserved PBMC in RPMI 1640 medium supplemented with 10% FBS, or RPMI 1640 medium supplemented with 10% FBS and 10 ng/mL recombinant human IL-15

dependent variables

NK cell subsets and phenotypes identified through surface and intracellular staining with fluorescently conjugated antibodies
Ab binding capacity (ABC) of perforin and granzyme within cells

control variables

Cryopreserved PBMC collected from 15 healthy normal adult donors
Viability marker (Fixable Aqua Dead Cell Stain Kit) used for staining
Staining panel based on the Optimized Multicolor Immunofluorescence Panel (OMIP-007)

controls

Quantum™ Simply Cellular® beads used to determine the Ab binding capacity (ABC) of perforin and granzyme within cells

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