Manufacturer’s instructions (Miltenyi Biotec). See also Chapter IV Section 1.4 Magnetic pre-enrichment for high-resolution detection and evaluation of uncommon cell populations. Intracellular staining: To analyze transcription factor 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 working with the Foxp3/ Transcription Factor Staining Buffer Set (eBioscience) as per the manufacturer’s instructions, following which, cells are stained for intracellular transcription components for 30 min or overnight. 1.8.four 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 Issue 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.five) 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 attainable. Having said that, as a consequence of distinct staining PPARĪ³ Activator Species circumstances, it might result in different staining intensities. CD24 Ab stainingEur J Immunol. Author manuscript; available in PMC 2020 July 10.Cossarizza et al.Pageis sensitive to EDTA. Distribution of iNKT cell subsets varies among organs as well as in between mouse strains. For instance, in liver iNKT1 cells constitute the predominant iNKT cell subset, whereas mesenteric lymph nodes predominantly contain iNKT2 cells [839]. In addition, BALB/c mice show a strong bias towards iNKT2 cells when compared to C57BL/6 mice [830]. 1.eight.6 Leading tricks iNKT cells are a rare population of T cells. Consequently, for some downstream analyses it can be advisable to perform enrichment using magnetic beads (see also Chapter IV Section 1.four Magnetic pre-enrichment for high-resolution detection and analysis of uncommon cell populations). We and other individuals have located that differences in frequencies of iNKT cells in mouse strains with iNKT cell deficiency, for example miR-181a/b-1-deficient mice, in comparison with wild-type mice are primarily retained upon enrichment by means of tetramers [840]. The underlying reason remains elusive but may very well be attributed to reduce affinity of tetramers when in comparison with Abantigen interaction. We and other individuals have employed Rag-GFP reporter mice to delineate developmental progression of iNKT cells in the thymus. Such a mouse model may perhaps assistance to further resolve NKT cell precursors and mature NKT cell PPARĪ± Agonist supplier populations inside the thymus [828, 841]. 1.eight.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 3 NKT1 NKT2 NKT17 CD44-NK1.1-CD24hiFSChi CD44-NK1.1-CD24loFSClo CD44+ NK1.1- CD44+NK1.1+ CD122+PLZFloT-bet+RORt- 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.