Manufacturer's directions (Miltenyi Biotec). See also Chapter IV Section 1.four Magnetic pre-enrichment for high-resolution detection

Manufacturer’s directions (Miltenyi Biotec). See also Chapter IV Section 1.four Magnetic pre-enrichment for high-resolution detection and analysis of rare cell populations. Intracellular staining: To analyze transcription element expression, magnetic-bead-enriched CD1d-PBS-57 tetramer+ cells from lymphoid organs are stained for surface markers and viability as described above. Samples are then fixed and permeabilized applying the Foxp3/ Transcription Factor Staining Buffer Set (eBioscience) as per the manufacturer’s directions, following which, cells are stained for intracellular transcription factors for 30 min or overnight. 1.8.4 MaterialsAuthor Manuscript Author Manuscript Author Manuscript Author Manuscript1.8.FCM buffer:PBS, three FCSRBC lysis buffer (Qiagen) Zombie Aqua Fixable Viability kit (Biolegend) anti-APC magnetic microbeads (Miltenyi Biotec) Foxp3/Transcription Element Staining Buffer Set (eBioscience) Tetramers: Mouse CD1d-PBS-57-APC (NIH tetramer core facility, Atlanta, USA) Unloaded mouse CD1d-APC (NIH tetramer core facility, Atlanta, USA) Antibodies: CD16/32 mAb (clone two.4G2) CD19 mAb (clone 6D5) Anti-B220 (clone RA3-6B2) CD11b mAb (clone M1/70) CD11c mAb (clone N418) Anti-TCR (clone H57-597) CD4 mAb (clone GK1.5) 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) Anti-CXCR3 (CD183, clone CXCR3-173) CD122 mAb (clone TM-b1)Pitfalls Simultaneous staining of cells with tetramer and anti-TCR is doable. On the other hand, due to distinct staining circumstances, it may result in different staining intensities. CD24 Ab stainingEur J Immunol. Author manuscript; accessible in PMC 2020 July 10.Cossarizza et al.Pageis sensitive to EDTA. Distribution of iNKT cell subsets varies amongst organs and also between mouse strains. For example, in liver iNKT1 cells constitute the predominant iNKT cell subset, whereas mesenteric lymph nodes predominantly contain iNKT2 cells [839]. Moreover, BALB/c mice show a strong bias towards iNKT2 cells when in comparison with C57BL/6 mice [830]. 1.eight.6 Leading tricks iNKT cells are a rare population of T cells. For that reason, for some downstream analyses it can be advisable to execute enrichment working with magnetic beads (see also Chapter IV Section 1.four Magnetic pre-enrichment for high-resolution detection and mGluR2 Agonist supplier evaluation of rare cell populations). We and other individuals have found that differences in frequencies of iNKT cells in mouse strains with iNKT cell deficiency, including miR-181a/b-1-deficient mice, in comparison with wild-type mice are primarily retained upon enrichment by means of tetramers [840]. The underlying cause remains elusive but might be attributed to reduce affinity of tetramers when in comparison to Abantigen interaction. We and others have employed Rag-GFP reporter mice to delineate developmental progression of iNKT cells within the thymus. Such a mouse model may well help to additional resolve NKT cell precursors and mature NKT cell populations inside the thymus [828, 841]. 1.8.7 Summary tableAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptMurine NKT cell population (TCR+CD1d-PBS-57/GalCer tetramer+)Phenotype/subphenotypeThymusStage 0 Stage 1 Stage 2 Stage three NKT1 NKT2 NKT17 CD44-NK1.1-CD24hiFSChi CD44-NK1.1-CD24loFSClo CD44+ NK1.1- CD44+NK1.1+ CD122+PLZFloT-bet+RORt- β-lactam Inhibitor Storage & Stability CD122-CD4+PLZFhiT-bet-RORt-PD-1+CCR7- CD122-CD4-PLZFintT-bet-RORt+PeripheryNKT1 NKT2 NKT17 CXCR3+PLZFloT-bet+RORt- CXCR3-CD4+PLZFhiT-bet-RORt- CXCR3-CD4-PLZFintT-bet-RORt+1.1.9.Mur.