Cd39 fitc

For cell phenotype analysis, monoclonal antibodies were directly added to 100 μL aliquots of heparinized whole blood and incubated at room temperature for 15 min. The antibodies used were: CD3 PECy7, CD14 APC, CD56 PE-Cy5 and CD19 PerCp (Biolegend, San Diego, CA, USA), CD4 Viogreen (Miltenyi Biotec GmBh, Bergisch Gladbach, Germany), CD39 Fitc, CD73 PE, CD25 BV421 and CD127 AlexaFluor 647 (BD Biosciences, San Jose, CA, USA). Red blood cells were lysed and white cells were fixed using BD FACS lysing solution (BD Bioscience). Negative populations were determined using respective anti-human isotype controls. Samples were acquired using a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec GmBh) and the data were analyzed using FlowJo software v. 10.0 (TreeStar Inc., Ashland, OR, USA).
We compared the frequencies of four subsets based on CD39 and CD73 expression (CD39⁻CD73⁺, CD39⁺CD73⁺, CD39⁺CD73⁻, and CD39⁻CD73⁻) within each studied population (B cells as CD3⁻CD19⁺, CD4⁺ T cells as CD3⁺CD4⁺, CD8⁺ T cells as CD3⁺CD4⁻, NK cells as CD3⁻CD4⁻CD19⁻CD56⁺, Tregs as CD3⁺CD4⁺CD25⁺⁺CD127⁻, and monocytes as CD14⁺). The percentage of these four subsets was related to their corresponding precursor populations (CD4⁺ T, CD8⁺ T, B, NK, and monocytes). In the case of Treg, the percentage was related to CD4⁺ T cells.

Single cell suspensions of leukocytes from blood, ascites, tumor or omental metastases were stained with the following monoclonal antibodies: anti-CD3 APC-H7 (BD Pharmingen, 560176), anti-CD4 eFlour 450 (eBioscience, 48-0048-42), anti-CD8 Pacific Orange (Invitrogen, MHCD0830), anti-CD25 APC (BD Pharmingen, 555434), or anti-CTLA-4 APC (BD Pharmingen, 555855), anti-CD28 PerCpCy5.5 (eBioscience, 45-0289-42), anti-CD38 PE-TR (Invitrogen, MHCD3817), anti-HLA-DR PE-Cy7 (BD Biosciences, 335795), anti-FoxP3 PE (eBioscience, 12-4777-42), anti-ki67 FITC (BD Pharmingen, 556026) or CD39 FITC (BD Pharmingen, 561444) or anti-Helios (Biolegend, 137214). Cells were stained in FACs buffer (1%FBS in PBS with 0.01%NaN₃) and fixed according to the ebioscience FoxP3 Fix-Perm kit protocol (eBioscience, 00-5521-00). All samples were run on a BD LSRII Flow cytometer and analysed by FACSDiva BD. Briefly, for every single flow cytometric antibody, we have used Fluorescent Minus One (FMO), to discriminate between positive and negative cells [35 (link)].

Four hours before stopping the culture, PMA (50 ng/mL) and ionomycin (1 μg/mL) (Sigma-Aldrich) were added. After incubation, the stimulated PBMCs were collected and IFNγ secretion was determined using an IFNγ secretion assay detection kit APC (Miltenyi Biotec GmBh). Additionally, the cells were stained with the following monoclonal antibodies: anti-human CD3-Viogreen (Miltenyi Biotec GmBh), CD8-PECy7, and CD25-APCy7 (Biolegend), CD39-Fitc and CD73-PE (BD Biosciences) as well as violet-fluorescent reactive dye (Thermofisher, Waltham, MA, USA). Samples were acquired with the MACSQuant Analyzer 10 flow cytometer, and the data were analyzed using FlowJo software.