Immunomagnetic selection

CD34⁺ hematopoietic progenitors from cord blood were isolated by immunomagnetic selection (StemCell Technologies Inc.) and cultured as previously described [40 (link)]. At day 6, cells were activated for 2h at 37°C with 100 ng/ ml of recombinant ligands (TNF-α 20 ng/ ml), washed and cultured for 36h in RPMI containing 10% FCS. When two treatments were successively applied, TEM were first exposed for 2h to a combined treatment of PlGF/TNF-α/ANG-2, washed and kept in culture for 30h in RPMI containing 10% FCS. They were then exposed to inhibitory treatments for 2h, washed and kept 24 hours longer in culture. TEM phenotype, cytokine secretion and pro-angiogenic activity were assessed by flow cytometry and in vivo or in vitro vascularization assay, respectively. Alternatively TEM were isolated from patient peripheral blood or tumor by CD14 immunomagnetic selection, exposed to treatments for 36h (30h exposure to angiogenic and inflammatory factors followed by 6h exposure to TIE-2 kinase inhibitor at 8 μM) and extensively washed. TEM viability was not affected under the different conditions of stimulation used and was > 95%

Naïve T cells from Foxp3‐Tocky mice were isolated by negative selection using immunomagnetic selection (StemCell Technologies) and 2 × 10⁵ cells cultured on anti‐CD3 (clone 1452C11, 2 μg/ml) and anti‐CD28 (clone 37.51, 10 μg/ml; both eBioscience)‐coated 96‐well plates (Corning) in the presence of 500 U/ml rhIL‐2 (Roche) and 5 ng/ml rhTGFβ (R&D) for 48 h in a final volume of 200 μl RPMI1640 (Sigma) containing 10% FCS and penicillin/streptomycin (Life Technologies). Cells were harvested then incubated with 100 μg/ml cycloheximide (Sigma) for the indicated time points before analysis by flow cytometry. For determination of the red half‐life, Blue⁻Red⁺ CD4⁺ T cells from Nr4a3‐Tocky mice were isolated by cell sorting and then cultured without TCR signals under the presence of rhIL‐2 for the indicated number of hours before analysis by flow cytometry. Importantly, IL‐2 signals do not induce Timer expression in Nr4a3‐Tocky T cells (Bending et al, 2018).