Immu 357

PBMCs or lymphocytes isolated from the colon or rectal mucosa were utilized for flow cytometry. Monoclonal antibodies against CD45 (clone J33; Beckman Coulter, Pasadena, CA, USA), CD3 (UCHT1, Beckman Coulter), CD4 (13B8.2; Beckman Coulter), CD8 (B9.11; Beckman Coulter), CD7 (8H8.1; Beckman Coulter), CD25 (B1.49.9; Beckman Coulter), CD30 (HRS4; Beckman Coulter), CD45RA (2H4; Beckman Coulter), CD56 (N901; Beckman Coulter), CD62L (DREG56; Beckman Coulter), CD127 (R34.34; Beckman Coulter), CCR4 (i.e., CD194; L291H4; BioLegend), HLADR (Immu-357; Beckman Coulter), and PD1 (CD279; PD1.3; Beckman Coulter) were employed. The immunostained cells were analyzed using FACScan (Navios flow cytometer, Beckman Coulter) and Kaluza analysis software (version 1.3; Beckman Coulter). Lymphocytes were separated by flow cytometry based on high CD45 antigen expression and forward and side scatter properties. Subsequently, the flow cytometry data were analyzed according to the percentage of cell populations detected in each quadrant on two-dimensional scatterplots. We calculated the percentages of CD4⁺, CD8⁺, CD56⁺, CD7⁺, PD1⁺, CCR4⁺, CD30⁺, and HLADR⁺ cells among CD3⁺ cells. We also assessed the percentages of Treg, CD45RA⁺, and CD62L⁺ cells among CD3⁺CD4⁺ cells and percentages of CD45RA⁺ and CD62L⁺ cells among CD3⁺CD4⁻ cells. In this study, we defined CD3⁺CD4⁺CD25⁺CD127^low/- cells as Treg cells.

As a second step, we measured the whole blood immunophenotyping of a subset of 15 FSHD patients and 15 new sex-age healthy controls. Samples were not pre-selected out of an initial pool. Peripheral fresh whole blood samples were analysed by flow cytometry as described by Aguirre-Gamboa et al. [28 (link)]. Briefly, samples were stained with a antibody cocktail containing the following fluorochrome conjugated antibodies (dilution, cone, distributor): CD45-KO (1:50, J33, Beckman Coulter), CD16-FITC (1:50, 3G8, Beckman Coulter), HLA-DR-PE (1:10, immu-357, Beckman Coulter), CD14-ECD (1:100, UCHT1, Beckman Coulter), CD4-PE-Cy5.5 (1:200, 13B8.2, Beckman Coulter), CD25-PC7 (1:50, M-A251, Becton & Dickinson), CD56-APC (1:50, N901, Beckman Coulter), CD8-APC-AF700 (1:400, B9.11, Beckman Coulter), CD19-APC-AF750 (1:50, J3-119, Beckman Coulter), CD3-PB (1:50, UCHT1, Beckman Coulter). Surface expression was measured on a 10-color Navios flow cytometer (Beckman Coulter) and analysed using Kaluza software version 1.3 (Beckman Coulter) (see Supplemental Figure 2 for gating strategy).