Invariant NKT (
i
NKT) cells are a small subset of thymus-generated T cells that produce cytokines to control both innate and adaptive immunity. Because of their very low frequency in the thymus, in-depth characterization of
i
NKT cells can be facilitated by their enrichment from total thymocytes. Magnetic-activated cell sorting (MACS) of glycolipid antigen-loaded CD1d-tetramer-binding cells is a commonly used method to enrich
i
NKT cells. Surprisingly, we found that this procedure also dramatically altered the subset composition of enriched
i
NKT cells. As such, NKT2 lineage cells that express large amounts of the transcription factor promyelocytic leukemia zinc finger were markedly over-represented, while NKT1 lineage cells expressing the transcription factor T-bet were significantly reduced. To overcome this limitation, here, we tested magnetic-activated depletion of CD24
+
immature thymocytes as an alternative method to enrich
i
NKT cells. We found that the overall recovery in
i
NKT cell numbers did not differ between these 2 methods. However, enrichment by CD24
+
cell depletion preserved the subset composition of
i
NKT cells in the thymus, and thus permitted accurate and reproducible analysis of thymic
i
NKT cells in further detail.
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