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. 2018 Feb 28;19(3):684. doi: 10.3390/ijms19030684

Figure 3.

Figure 3

GM-CSF-primed neutrophils remain sensitive to FASL killing. (a) Primary WT and Bid−/− neutrophils were primed with GM-CSF (1 ng/mL) for 30 min prior to stimulation with FASL (100 ng/mL) for indicated time points. Viability was assessed by flow cytometry. n ≥ 3, mean ± SEM. (b) Primary WT and Bid−/− neutrophils were primed with GM-CSF (1 ng/mL) for 30 min and pre-treated with either Q-VD-OPh (20 μM), Nec.1 (20 μM) or the mouse MLKL inhibitor GW806742X (1 μM) for 30 min followed by stimulation with FASL (100 ng/mL) for indicated time points. Viability was assessed by flow cytometry. n ≥ 3, mean ± SEM. (c) In vitro differentiated Ripk3−/− neutrophils were primed with GM-CSF (1 ng/mL) for 30 min prior to stimulation with FASL (100 ng/mL) with or without Q-VD-OPh (20 μM) for indicated time points. Viability was assessed by flow cytometry. n ≥ 3, mean ± SEM. (a–c): p < 0.05 (*), p < 0.01 (**), p < 0.005 (***) and p < 0.001 (****). (d) In vitro differentiated WT and Bid−/− neutrophils were primed with GM-CSF (1 ng/mL) for 30 min and subsequently treated with FASL (100 ng/mL) for 0–8 h. Lysates were assessed by immunoblotting. Presented immunoblots are representative of at least two independent experiments.