Cd73 pe clone ad2

The expression of ectonucleotidases (CD39 and CD73) on αβDNT and activated Treg cells was evaluated by flow cytometry. The gating strategy for activated Treg cells was performed as previously described (5 (link)). PBMCs were stained with directly conjugated antibodies for 30 min at 4°C in the dark. The cells were washed before flow cytometry analysis. Antibodies used included anti-human CD3-BV785 (clone SK7), CD4-APC-fire750 (clone SK3), αβTCR-BV421 (clone IP26), CD56-BV510 (clone HCD56), CCR7 (CD197)-PE-cy7 (clone G043H7), HLA-DR-AF700 (clone L243), CD73-PE (clone AD2), CD39-BV605 (clone A1, BioLegend, San Diego, CA, USA), CD45RA-BV711 (clone HI100), CD38-BUV737 (clone HB7), CD25-PE-CF594 (clone M-A251), CD8-FITC (clone SK1), CD8-BUV395 (clone RPA-T8, BD Biosciences, San Diego, CA, USA), and the corresponding isotype controls. Data were acquired on a BD LSR Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR, USA).

Patients with severe aortic stenosis underwent surgical heart valve replacement. Primary interstitial cells were obtained from the leaflets of the excised heart valves, isolated by using collagenase, and cultured until confluent monolayer as it is previously described (Kostina et al., 2018 (link); Malashicheva et al., 2018 (link)). Cells obtained after passages 2–4 were used in further experiments. For this, 12-well plates were seeded with 160,000 cells/well followed by incubating with adenosine (30 mmol) or ATP (100 mmol) 24 h later according to Mahmut et al. (Mahmut et al., 2015 (link)). Control cells were unstimulated. Cell phenotyping was performed on days 1, 3, and 7.
Cell staining with anti-human CD39-FITC (clone A1, cat. 328206, BioLegend, Inc., USA) and CD73-PE (clone AD2, cat. 550257, BD Pharmingen™, USA) was performed in accordance with the manufacturer’s recommendations followed by collecting the data on flow cytometer Guava EasyCyte 8 (Millipore, USA).
Mean intensity fluorescence (MIF) for CD39- and CD73-positive staining was analyzed by comparing it with the intensity of isotype-match control antibodies conjugated with relevant fluorochrome followed by estimating the percentage of T-cell subsets expressing CD39−CD73−, CD39−CD73+, CD39+CD73+, and CD39−CD73−.