Neutrophils were cultured at 5 million cells/mL for 2-20 h at 37°C, in normoxia (21% O2, 5% CO2) or hypoxia (1% (3kPa) O2, 5% CO2) in two types of pre-equilibrated RPMI media 1640 with 10% fetal bovine serum (FBS) (GIBCO) and 1% Penicillin/Streptomycin, differing in the presence or absence of glucose. In the absence of glucose, dialyzed glucose-free FBS was used. To study the effects of stimulation and metabolic inhibitors, neutrophils were cultured with (100ng/mL) LPS E.coli serotype R515 (Enzo), CP-91149 (Sigma-Aldrich), 2-deoxyglucose (Sigma-Aldrich), Oligomycin A (Sigma-Aldrich) and Etomoxir (10uM) (CNIO Carlos III Therapies) (Raud et al., 2018 (link); Yao et al., 2018 (link)), Fructose-1,6-bisphosphatase-1 Inhibitor (FBP1i) (Cambridge Bioscience), MB05032 (Tocris Bioscience), 3-Mercaptopicolinic acid (HCl) (3-MPA) (Cambridge Bioscience), Glutaminase Inhibitor II, BPTES (MERCK Chemical).
Corresponding Organization : University of Edinburgh
Other organizations :
VIB-KU Leuven Center for Cancer Biology, University of Sheffield, MRC Institute of Genetics and Molecular Medicine, Roslin Institute, Centro de Investigación Biomédica en Red Diabetes y Enfermedades Metabólicas Asociadas, Institute for Research in Biomedicine, Universitat de Barcelona, Edinburgh Cancer Research, KU Leuven, Sun Yat-sen University
Treatment with Fructose-1,6-bisphosphatase-1 Inhibitor (FBP1i)
Treatment with MB05032
Treatment with 3-Mercaptopicolinic acid (3-MPA)
Treatment with Glutaminase Inhibitor II, BPTES
dependent variables
Cell response (not explicitly specified)
control variables
Cell density (5 million cells/mL)
Incubation time (2-20 h)
Temperature (37°C)
Culture media (RPMI 1640 with 10% FBS and 1% Penicillin/Streptomycin)
CO2 level (5% CO2)
positive controls
Normoxia (21% O2, 5% CO2)
negative controls
Absence of glucose in media
Absence of stimuli and metabolic inhibitors
Annotations
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