Purification and Sequencing of iNKT Cells

Primary human iNKT cells were initially purified from PBMCs or thymi using anti-iNKT MicroBeads (clone 6B11, Miltenyi Biotec, Auburn, CA). Subsequently, the cells were stained with anti-Vα24 (clone C15) and anti-Vβ11 mAbs. The double positive population was sorted with a FACSAria (BD Biosciences, Mississauga, Canada). The purity of the sorted cells was consistently >95%. TCRβ sequencing was performed at Adaptive Biotech using the ImmunoSEQ platform (Seattle, WA). This method was used to capture the frequencies of individual TCRs in biologic samples with accurate reproducibility and a sensitivity of 1/100,000 T cells (26 (link)).

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Chamoto K., Guo T., Scally S.W., Kagoya Y., Ancruzowski M., Wang C.H., Rahman M.A., Saso K., Butler M.O., Chiu P.P., Julien J.P, & Hirano N. (2016). Key residues at third CDR3β position impact structure and antigen recognition of human invariant natural killer T cell receptors. Journal of immunology (Baltimore, Md. : 1950), 198(3), 1056-1065.

Publication 2016

anti-iNKT MicroBeads FACSAria

Adaptive Biologic Clone Human Inkt cells Mabs Microbeads Sensitivity T cells Tcrs Tcrβ Thymi

Corresponding Organization : University of Toronto

Other organizations : Hospital for Sick Children

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Variable analysis

independent variables

Method used to purify primary human iNKT cells from PBMCs or thymi (using anti-iNKT MicroBeads, clone 6B11)
Staining of cells with anti-Vα24 (clone C15) and anti-Vβ11 mAbs
Cell sorting using a FACSAria (BD Biosciences)

dependent variables

Purity of sorted cells (consistently >95%)
Frequencies of individual TCRs in biological samples

control variables

Not explicitly mentioned

controls

No positive or negative controls were explicitly mentioned in the protocol.

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