Manufacturer’s guidelines (Miltenyi Biotec). See also Chapter IV Section 1.4 Magnetic pre-enrichment for high-resolution detection and evaluation of rare 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 applying the Foxp3/ Transcription Factor Staining Buffer Set (eBioscience) as per the manufacturer’s directions, PDGF-BB Proteins supplier 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) Persephin Proteins Recombinant Proteins anti-APC magnetic microbeads (Miltenyi Biotec) Foxp3/Transcription Aspect 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 2.4G2) 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. However, because of distinct staining circumstances, it may lead to distinctive staining intensities. CD24 Ab stainingEur J Immunol. Author manuscript; out there in PMC 2020 July ten.Cossarizza et al.Pageis sensitive to EDTA. Distribution of iNKT cell subsets varies between organs and also among mouse strains. As an example, 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 sturdy bias towards iNKT2 cells when when compared with C57BL/6 mice [830]. 1.eight.six Leading tricks iNKT cells are a uncommon population of T cells. As a result, for some downstream analyses it is advisable to execute enrichment applying magnetic beads (see also Chapter IV Section 1.4 Magnetic pre-enrichment for high-resolution detection and evaluation of uncommon cell populations). We and others have found that variations in frequencies of iNKT cells in mouse strains with iNKT cell deficiency, such as miR-181a/b-1-deficient mice, in comparison to wild-type mice are basically retained upon enrichment via tetramers [840]. The underlying reason remains elusive but could be attributed to decrease affinity of tetramers when in comparison to Abantigen interaction. We and other folks have employed Rag-GFP reporter mice to delineate developmental progression of iNKT cells within the thymus. Such a mouse model may well assist to additional resolve NKT cell precursors and mature NKT cell 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 three 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.