Anti cd127 alexafluor647 clone hil 7r m21

Fluorochrome-conjugated monoclonal antibodies were used for PBMC staining, including anti-CD4 FITC (clone RPA-T4), anti-CD8 PE (clone HIT8α), anti-CD25 PE-Cy5 (clone M-A251), anti-CD127 AlexaFluor647 (clone HIL-7R-M21) (BD Bioscience). Stained samples were run on flow cytometer and analysed using FlowJo software. Initially, lymphocytes were distinguished on the basis of their FSC and SSC properties. Next, populations of CD4+ (Th, helper T cells) and CD8+ (Tc, cytotoxic T cells) lymphocytes were delineated. The expression of CD25 (IL-2Ra; activation marker) and CD127 (IL-7R; development-related marker) were assessed within the gated subsets of lymphocytes (Figure S1D). In addition, regulatory T cells (Tregs) were gated and analysed in the context of the frequency within the lymphocytes. The gating strategy for Tregs was based on the presence of a high CD25 expression and low/no CD127 on T cells’ surface (Figure S1E). Unstained and FMO controls were used for setting gating strategies (Figure S3A–D).

To determine the Treg profile and function, 1x10⁶ PBMCs were incubated for 15 min at room temperature with the anti-CD3-PerCP-Cy™5.5 (clone UCHT1), anti-CD4-APC-H7 (clone SK3), anti-CD8-Alexa Fluor^® 700 (clone RPA-T8), anti-CD25-PE-CF594 (clone M-A251), anti-CD127-Alexa Fluor^® 647 (clone HIL-7R-M21), anti-CD45RA-PE-Cy™7 (clone HI100), anti-CD39-BV650 (clone TU66), and anti-CD73-BV421 (clone AD2) monoclonal antibodies (all from BD Biosciences). Cells were then washed and fixed/permeabilized (eBioscience, catalog no. 00-5521-00) using FOXP3 staining buffer (eBioscience, catalog no. 00-5523-00), and finally stained with the combination of conjugated antibodies anti-Ki67-FITC (BD Biosciences, catalog no. 556026) and anti-FOXP3-PE (BD Biosciences, clone 259D/C7) for nuclear epitope staining. Next, cells were washed with PBS, fixed with 1% formaldehyde in PBS (Sigma-Aldrich, catalog no. 1004960700) for 15 min at room temperature and analyzed by flow cytometry.

Lymphocytes present in PBMC (BC group) and ascites (ASC and ASC-CA groups) were phenotyped for the identification of their subsets. A flow cytometric-based assay was used according to standard procedures [37 (link)]. Briefly, the cells were mixed with 50 µL of staining solution containing a mix of fluorochrome-conjugated monoclonal antibodies at a 1:50 dilution; anti-CD3 APC-Cy7 (clone SK7), anti-CD4 PerCP-Cy5.5 (clone RPA-T4), anti-CD25 PE (clone M-A251), anti-CD56 PE-Cy7 (clone B159), anti-CD127 Alexa Fluor647 (clone HIL-7R-M21) (BD Pharmingen™), and anti-CD8 FITC (clone OKT8) (Miltenyi Biotec). Cells were incubated for 30 min on ice and protected from light. After the incubation, cells were washed twice with PBS and the final pellets suspended for acquisition in a FACSVerse cytometer using the FACSuite software (Becton Dickinson, San Jose, CA, USA). FlowJo software was used for the data analysis. The lymphocyte population was identified by the FSC and SSC parameters, and then FSC-Area vs. FSC-Height was used to eliminate doublets. Within the lymphocyte populations, the CD3⁺ lymphocyte population was identified by anti-CD3 APC-Cy7. Within the CD3⁺ lymphocytes, CD4⁺ and CD8⁺ populations were distinguished. Within the CD4⁺ population, the T-reg population was quantified by the parameters anti-CD25 PE vs. anti-CD127 Alexa Fluor647.