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OMIP-062

A 14‐Color, 16‐Antibody Panel for Immunophenotyping Human Innate Lymphoid, Myeloid and T Cells in Small Volumes of Whole Blood and Pediatric Airway Samples

Dawid Swieboda, Yanping Guo, Sophie Sagawe, Ryan S. Thwaites, Simon Nadel, Peter J.M. Openshaw, Fiona J. Culley Cytometry PART A, Volume 95, Issue 12, 1231-1235 (2019)

PURPOSE: This 14‐color, 16‐antibody OMIP was designed for enumeration of leukocyte responses in pediatric samples, where sample volumes and cell numbers can be very low. Leukocytes identified by this panel include all major members of the innate lymphoid cell (ILC) family (ILC1s, ILC2s, and ILC3s), natural killer cells (NK cells), granulocytes (neutrophils and eosinophils), T‐cells (CD4+ and CD8+), mucosal‐associated invariant T cells (MAIT cells) and NKT‐like cells. The protocol was optimized using small volumes of peripheral blood and validated in airway samples obtained from children (< 2 years of age) admitted to a pediatric intensive care unit (PICU). Given this backdrop, this OMIP is widely applicable to clinical research using low volume or paucicellular samples, such as studies of innate and adaptive immune responses in infants and children, with potential clinical application in diagnostics and monitoring of patients by pediatricians.

CELL TYPE: Whole peripheral blood, cord blood, PBMCs, nasal aspirate, and tracheal aspirate samples

MACHINE: BD LSR Fortessa

Marker Target Clone Fluorochrome Purpose
CD127 Human A019D5 Brilliant Violet 421 ILCs 
CD14 Human 63D3 Brilliant Violet 510 Lineage  
CD19 Human HIB19 Brilliant Violet 510 Lineage  
Fc epsilon R1 alpha Human Brilliant Violet 510 Lineage  
CD123 Human 6H6 Brilliant Violet 510 Lineage  
CD4 Human RPA-T4 Brilliant Violet 605 CD4+ T cells 
CD16 Human 3G8 Brilliant Violet 650 NK cells/neutrophils 
CD8 Human SK1 Brilliant Violet 711 CD8+ T cells 
TCR V alpha 7.2 Human 3C10 Brilliant Violet 785 MAIT cells 
CD45 Human HI30 FITC Leukocyte
CD117 Human A3C6E2 PerCP-Cy5.5 ILC3  
CD3 Human OKT3 PE T cells 
CD161 Human HP-3G10 PE-Dazzle 594 MAIT cells 
CD56 Human 5.1H11 PE-Cy7 NK/NKT-like cells 
CD294 Human BM16 Alexa Fluor 647 ILC2/Th2/Tc2 subsets 
CD66b Human G10F5 Alexa Fluor 700 Eosinophils 
Dead cells Human Live/Dead Fix Near IR (775) Viability