Rting gates were set as depicted in Additional file 2: Figure S
Rting gates were set as depicted in Additional file 2: Figure S2 using the FMOs controls, as previously described [21,101]. The viability staining Vivid (Invitrogen) was included in each staining cocktail to exclude dead cells. The post-sort quality analysis indicated that sorted subsets were in average >99 pure based on CD3, CD4 and CD45RA expression (Additional file 1 and 2: Figure S1 2). Sorting based on CD25/CD127 expression led to a significant enrichment of naive T-cell subsets (>80 ), with the exception of CD25+CD127+ which were typically >50 enriched (Additional file 2: Figure S2).Th17 polarization in vitroNaive and memory T-cell subsets (106 cells/ml) sorted by FACS were stimulated with immobilized CD3 and soluble CD28 Abs (1 g/ml) and cultured in RPMI 1640 media supplemented with human recombinant IL-23 (50 ng/ml), TGF- (10 ng/ml), IL-1 (10 ng/ml), and IL-6 (50 ng/ml) cytokines and neutralizing IL-4 (1 g/ml) and IFN- Abs (10 g/ml) (R D Systems) for 12 days. Media including polarizing cytokines, Abs, and IL-2 (5 ng/ml) was refreshed at day 4 and 8 post-culture. Cells were split at day 4 and/or 8 post-culture for an optimal density ONO-4059 cancer 1-2×106 cells/well.Intracellular staining for flow cytometry analysisTotal or memory CD4+ T-cells were PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28494239 sorted from frozen PBMCs by negative selection using magnetic beads (Miltenyi). Cell purity (typically >95 ) was determined by FACS analysis upon staining with CD3-PB, CD4-Alexa700, and CD8-FITC Abs for total T-cell enrichment, together with CD45RA-APC eFluor780 and CCR7-PeCy7 Abs for memory T-cell enrichment. In some experiments (Figure 1),Cells were stimulated with PMA (50 ng/ml) and Ionomycin (1 g/ml) in the presence of Brefeldin A (2 g/ml) for 5 h or 17 h. The intracellular expression of IL-17A, IFN- and TNF- was quantified by flow cytometry (BD LSRII) upon staining with appropriate Abs using the Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s protocols. The intracellular expression of FoxP3 was quantified using the Anti-Human Foxp3 Staining Set Alexa Fluor?488 (eBioscience) according to the manufacturer’s protocol.ELISA quantification of IL-17A productionIL-17A levels in cell culture supernatants were quantified by a specific ELISA assay (eBiosciences) according to the manufacturer’s protocol.DaFonseca et al. Retrovirology (2015) 12:Page 20 ofQuantitative SYBR green real-time RT-PCRTotal RNA was isolated using RNeasy kit (Qiagen). The quality (260/280 ratio) and quantity of RNA collected were measured by a Pearl nanophotometer (Implen, Munich, Germany). One step SYBR Green real-time RTPCR (Qiagen) was carried out in a LightCycler 480 II (Roche) according to the manufacturer’s recommendations. The quantification of RORC mRNA relative to the 28S rRNA levels was performed as we previously described [21]. Each RT-PCR reaction was performed in triplicates.Real-time PCR quantification of Gag and integrated HIVDNAAdditional file 2: Figure S2. Flow cytometry sorting of phenotypically naive CD4+ T-cells with differential expression of CD25 and CD127. Total CD4+ T-cells were isolated from PBMCs by negative selection using magnetic beads (Miltenyi). Cells were PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/29072704 stained with a cocktail of CD3, CD4, CD45RA, CCR7, CD25, and CD127 Abs and the viability dye Vivid. Viable (Vivid-) naive-like (CD45RA+CCR7+) CD4+ T-cells with a CD25+CD127- (nTregs), CD25-CD127+ (conventional nT), CD25+CD127+ (DP, double positive), and CD25-CD127- (DN, double negative) phenotype were sorted by flow cytometry.

Leave a Reply