Supplementary MaterialsSupplementary Shape 1 MACS-mediated enrichment of thymic manipulations (16,17,18,19,20). em i /em NKT cells that express the canonical V14-J18 TCR (15,21). In the thymus of BALB/c mice, usually less than 1% of total thymocytes correspond to CD1dTet+ em i /em NKT cells (Fig. 1A top) (1,8). Notably, not all of them are functionally mature, and a significant proportion of these cells correspond to immature pre-selection CD24hi em i /em NKT cells (Fig. 1A, bottom). Because of their scarcity, enrichment of thymic em i /em NKT cells greatly facilitates their subsequent analysis. To this end, MACS-based positive selection of CD1d+ em i /em NKT cells from total thymocytes is frequently employed to increase frequencies of em i /em NKT cells and to facilitate detailed interrogation of their phenotype and function (16,17,18,19,20). We confirmed that this protocol indeed dramatically enriched for em i /em NKT cells (Fig. 1B), usually resulting in an approximately 9018.7-fold increase in em i /em NKT cell frequencies (Fig. 1C). The flow-through fraction of MACS columns, on the other hand, demonstrated reduced frequencies of em i /em NKT cells significantly, indicating preferential binding of MACS-bead tagged em i /em NKT cells to magnetized MACS columns (Supplementary Fig. 1A). Oddly enough, we also observed a dramatic change in TCR surface area appearance and in the quantity of Compact disc1dTet+ binding by post-enrichment em i /em NKT cells (Fig. 1D). In comparison to pre-enrichment em we /em NKT cells, MACS-selected em we /em NKT cells portrayed greater levels of TCR and demonstrated elevated staining for Compact disc1dTet reagents (Fig. 1D). These outcomes suggested that Compact disc1dTet-mediated retention of em i /em NKT cells in MACS columns gets the unintended aftereffect of enriching for em i /em NKT cells with bigger amount of surface area TCR appearance and greater Compact disc1dTet-binding capacity. Along these relative Almorexant lines, we discovered that the unselected flow-through small fraction included few em i /em NKT cells still, but they portrayed much small amounts of TCR and demonstrated reduced binding of Compact disc1dTet (Supplementary Fig. 1B). Hence, Compact disc1dTet-binding MACS columns become a mobile sieve which preferentially enriches for em i /em NKT cells that bind better amounts of Compact disc1dTet. Collectively, these outcomes indicated that MACS-based collection of Compact disc1dTet+ cells presents a bias through the enrichment of em i /em NKT cells, in order that em i /em NKT cells expressing higher degrees of surface area TCR are preferentially maintained. Open in another window Body 1 Compact disc1d-tetramer-based enrichment of thymic em i /em NKT cells. (A) Id of em i /em NKT cells in BALB/c thymocytes by Compact disc1d tetramer (Compact disc1dTet) vs. TCR (best) or Compact disc1dTet vs. Compact disc24 evaluation (bottom level). Email address details are representative of 5 indie tests. (B) MACS-based enrichment of Compact disc1dTet+ em i /em NKT cells is certainly demonstrated by Compact disc1dTet vs. TCR (best) or Almorexant Compact disc1dTet vs. Compact disc24 evaluation (bottom level) of em i /em NKT cells in total thymocytes or after MACS column enrichment. Results are representative of 5 impartial experiments. (C) Percentages of em i /em NKT cells in total thymocytes (before) and CD1dTet-enriched fraction (after). Plot shows summary of 5 impartial experiments. (D) Surface TCR expression and CD1dTet staining on thymic em i /em NKT cells before and after MACS-mediated enrichment for em i /em NKT cells. Histograms (left) are representative and graphs (right) show summary of 5 impartial experiments. (E) Intranuclear staining for PLZF and RORt shows subset distribution before and after MACS-mediated enrichment for thymic em i /em NKT cells. Enriched em i /em NKT cells were stained for CD24 and gated on CD24lo to identify mature em i /em NKT cells. Dot plots (left) are representative and graphs (right) show summary of 5 impartial experiments.NS, not significant. **p 0.01; ***p 0.001 were considered statistically significant. The amount of surface TCR and binding of CD1dTet differ Almorexant Rabbit Polyclonal to RRM2B among individual em i /em NKT subsets (25). Thus, we wished to examine if MACS-based em i /em NKT enrichment would also skew the subset composition of enriched em i /em NKT cells, when compared to that of pre-enrichment em i /em NKT cells. Individual em i /em NKT subsets can be identified by the distinct expression of 3 transcription factors, namely PLZF, RORt, and T-bet (9,26). NKT1 cells express low amounts of PLZF but high levels of T-bet. NKT2 cells, on the other hand, are abundant for PLZF but not for RORt or T-bet. Finally, NKT17 cells express the signature transcription factor RORt, and they are absent for T-bet (9,27). Here, we found that MACS-enrichment for CD1dTet+ cells induced a significant bias for NKT2 lineage cells, with a concomitant loss in NKT1 cells (Fig. 1E). The unbound fraction of em i /em NKT cells that were recovered from the flow-through of the MACS column, on the other hand, showed a reverse enrichment for TCRlo NKT1 lineage cells and selective loss of TCRhi NKT2 cells (Supplementary Fig. 1C). Collectively, these results unveil a previously unappreciated pitfall in MACS-based em i /em NKT enrichment protocols that skews the subset composition of recovered em i /em NKT cells into NKT2 lineage cells. Because of this limitation, it was found by us necessary to develop an alternative method of enrich em i /em NKT cells, and to create a method that could not make use of anti-TCR or Compact disc1dTet for enrichment. Magnetic-activated depletion of Compact disc24+ thymocytes permits effective em /em NKT cell enrichment Compact disc24 we.