Ol. Author manuscript; accessible in PMC 2020 July ten.Cossarizza et al.PageAuthor Manuscript1.9.five PitfallsCD11b mAb (clone M1/70) CD11c mAb (clone N418) Anti-TCR (clone H57-597) Anti-NK1.1 (clone PK136) CD44 mAb (clone IM7) CD24 mAb (clone M1/69) Anti-PLZF (clone Mags.21F7) Anti-T-bet (clone O4-46) Anti-RORt (clone Q31-378 or B2D)MAIT cells constitute an very rare cell population, rendering subset evaluation prone to errors based on background staining (see Chapter V Section 1 Sigma 1 Receptor Modulator site uncommon cells–General guidelines). This difficulty is exacerbated in the analysis of genetically modified mice with developmental defects within the MAIT cell lineage. To minimize background, it really is pivotal to involve lineage markers inside a dump channel and/or enrich before downstream analysis. B cells in unique show a high degree of nonspecific binding of your MR1 tetramer (both 5OP-RU and 6-FP loaded). Simultaneous staining of cells with tetramer and anti-TCR is probable. Nonetheless, due to distinct staining conditions it may result in diverse staining intensities. CD24 antibody staining is sensitive to EDTA. 1.9.six Top tricks To be able to overcome issues related with low frequencies of MAIT cells, it is actually frequently advised to enrich for MR1-OP-RU-tet+ cells for subset analysis whenever feasible; see also Chapter IV Section 1.four Magnetic preenrichment for high-resolution detection and evaluation of uncommon cell populations. Notably, it has been demonstrated that magnetic-bead-based enrichment via tetramers essentially retains differences between wildtype frequencies and reduced MAIT-cell frequencies observed in genetically modified mice [841, 847]. The underlying mechanism remains unclear, but can be connected to the relative inefficiency of tetramer-based enrichment, which in turn could be as a consequence of decrease affinity of tetramer when compared to antibody-mediated binding. Furthermore, it is certainly crucial to exclude non-T lineage cells, most notably B cells, through gating to limit background staining. It truly is also advisable to incorporate nonbinding MR1-FP tetramers as background controls. Finally, for precise quantitation of MAIT cells, dual tetramer staining making use of a combination of MR1-OP-RU-APC and PE labeled tetramers may perhaps assist to minimize background [841]. We and others have employed Rag-GFP PDE5 Inhibitor supplier reporter mice to delineate developmental progression of MAIT cells in the thymus. Such a mouse model may perhaps assistance to further resolve MAIT cell precursors and mature MAIT cell populations within the thymus [828, 841].Author Manuscript Author Manuscript Author ManuscriptEur J Immunol. Author manuscript; readily available in PMC 2020 July 10.Cossarizza et al.Page1.9.Summary tableAuthor Manuscript Author Manuscript Author Manuscript1.ten.Murine MAIT cell population (Lin-TCR+MR1-5-OP-RU tetramer+)Phenotype/subphenotypeThymusstage 1 stage 2 stage three MAIT1 MAIT17 CD24+CD44-CCR7-PLZF- CD24-CD44-CCR7+PLZF- CD24-CD44+CCR7-PLZFhi T-bet+RORtlo T-bet-RORthiPeripheryMAIT1 MAIT17 T-bet+RORtlo T-bet-RORthi1.1.10.Murine intestinal intraepithelial T cellsOverview In this section, we describe protocols to isolate and analyze murine intestinal intra-epithelial lymphocytes (iIELs) and lamina propria lymphocytes (LPLs) by FCM. In particular, the protocol iIEL isolation and most of the subsequent flow cytometric evaluation applies similarly to and iIELs, which are extremely equivalent cell kinds.1.ten.Introduction The intestinal epithelium constitutes among the list of greatest surface barriers in mammals and is in continuous make contact with together with the (gut l.
