Pt Author Manuscript Author Manuscript17.9.3.1 Murine polyclonal suppression assay: Step-by-step sample preparation (see Table 15

Pt Author Manuscript Author Manuscript17.9.3.1 Murine polyclonal suppression assay: Step-by-step sample preparation (see Table 15 for reagents) Single cell suspensions of lymph nodes or spleen of a Foxp3-GFP reporter mouse are subjected to Osteoprotegerin Proteins medchemexpress damaging collection of CD4 T-cells by magnetic beads (CD4+ T Cell Isolation Kit, Miltenyi Biotec). Cells are then stained for 30 min at 4 with Abs for CD3, CD4, CD25, and B220 and sorted on a BD Aria-II. Tregs are sorted as CD3+CD4+B220-Foxp3+CD25+ and confirmed to possess a post sort purity of 90 + (Fig. 73A). Conventional T (Tconv) cells are sorted as CD3+CD4+B220-Foxp3-CD25-. When a Foxp3 reporter mouse is just not available CD25 and GITR could be used in its place (Fig. 73B). CD4 Tconv are then stained with 1 M CFSE for ten min in serum totally free media at room temperature. Excess CFSE is then quenched by addition of media+10 FCS just before washing three occasions. A total of 1 104 Tconv cells per effectively are cultured with or with no Treg cells at varied ratios (0:1, 1:1, 1:two, 1:4, 1:8 Treg:Tconv) for three days within the presence of 105 -irradiated CD4 depleted APCs (18.5Gy irradiated CD4 depleted splenocytes obtained by magnetic separation in step 1) and 1 g/mL soluble CD3 mAb (Clone: 145C11) in 96-well Ubottomed plates, in RPMI media containing ten FCS, 2-ME, L-glutamine and Penicillin/ streptomycin with a final volume of 200 L. In all situations the number of Tconv is fixed when the number of Tregs is changed to get the intended ratios. In the end of your 3 day culture period, cells are then stained with CD4 mAb, CD25 mAb, and IR Live/Dead dye and data collected on a BD LSR Fortessa. 17.9.3.2 Human polyclonal suppression assay: Step-by-step sample preparation (see Table 16 for reagents)Eur J Immunol. Author manuscript; obtainable in PMC 2020 July 10.Cossarizza et al.PageInitially PBMCs are isolated from fresh blood via Ficol aque centrifugation in Leucosep tubes. CD4 T-cells are enriched by negative collection of CD4 cells with magnetic beads (Miltenyi). Cells are stained with Abs for CD4, CD45RA, CD127, and CD25 for 30 min at 4 . Bulk Treg cells is usually sorted as CD3+CD4+CD127loCD25+ (Fig. 73C). If finer fractionation of Treg cells is needed, CD127loCD25+ cells can then be further separated into fraction I Na e Tregs, fraction II effector Tregs and fraction III non-suppressive cells (Fig. 73C) [675]. It needs to be noted that whilst fraction III as a complete is mostly created up of Foxp3 expressing non-Treg cells it might contain 200 CXCR5+ effector Tfr, which are functionally suppressive Treg cells [676]. Na e responder Tconv cells are sorted as CD25-CD45RA+CD4+CD3+ after which stained with 1 M CFSE. A total of 1 104 Tconv cells are co-cultured with different ratios of Tregs cells (0:1, 1:1, 1:2, 1:four, 1:eight Treg:Tconv) and 1 105 -irradiated APC (18.5Gy irradiated CD4 depleted PBMCs obtained by magnetic separation in Step 1) and stimulated with 1 g/mL soluble CD3 mAb (Clone: OKT3) for 4 days in 96-well round-bottom plates in RPMI medium containing 10 AB serum, 2-ME, L-glutamine, HEPES, and penicillin/ streptomycin in a final volume of 200 L. In all situations the number of Tconv and APC is fixed though the number of Tregs is changed to acquire the intended ratios. After a culture Integrin alpha X Proteins Purity & Documentation period of 4 days cells were then stained with CD4, CD25 and IR Live/ Dead dye and data collected on a BD LSR Fortessa. 17.9.four Suppression assays and antigen-specific T cellsAuthor Manuscript Author Manuscript Author Manuscript Author Manuscript17.9.4.1 Human suppression assa.