Spectral flow cytometer

Cell preparations from spleen, thymus, lymph nodes, liver, epididymal fat pads, lung, or Peyer's patches were harvested and exposed to hypotonic lysis to erythrocytes. Following cell preparation, cells were stained and analyzed using a BD LSRFortessa and a Sony Spectral Flow Cytometer. CD1d‐PBS57 (CD1d‐αgal) tetramers were obtained from the NIH Tetramer Core Facility. The following antibodies used for staining were obtained from BioLegend: IFNγ (Clone XMG1.2, Cat 505830), IL‐4 (Clone 11B11, Cat 504109), T‐bet (Clone 4B10, Cat 644816), CD3ε (Clone 17A2, Cat 100241), GL7 (Clone GL7, Cat 144609), B220 (Clone RA3‐6B2, Cat 103243), IgG1 (Clone RMG1‐1, Cat 406610), IgG2b (Clone RMG2b‐1, Cat 406707), and IgD (Clone 11‐26c.2a, Cat 405711). The following antibodies were from eBioscience: RORγt (Clone B2D, Cat 17‐6981‐80) and PLZF (Clone Mags.21F7, Cat 53‐9320‐82). The following antibody is from BD Pharmingen: IgA (Clone C10‐3, Cat 559354).

Starting form fresh peripheral blood mononuclear cells (PBMCs), two different subsets of CD4 T-cells were purified: resting memory CD4 T (Trm) cells (defined as CD4+CD45RO+CD25−CD69−HLADR−) and peripheral T follicular helper (pTfh) cells (defined as CD4+ CD45RO+ CXCR5+). For this purpose, immunomagnetic separation was employed as has been described elsewhere^{47 (link)}. Purity of each CD4 subset was tested by flow cytometry using a Sony Spectral flow cytometer. Median [Interquartile range] purity for the Trm cells in the whole population of subjects analyzed was 95%[93–97%] and for the pTfh cells was 92%[90–93%].