Live h1n1 attenuated influenza a pr8 iav

TruCulture whole blood stimulations were performed as previously described.¹⁵ (link) Briefly, tubes were prepared in batch with the indicated stimulus, resuspended in a volume of 2 mL buffered media, and maintained at −20°C until time of use. Stimuli in this study included lipopolysaccharide (LPS, 10 ng/mL) derived from E. coli O111:B4 (Invivogen), vaccine-grade poly I:C (pIC, 20 μg/mL) (Invivogen), live Bacillus Calmette-Guerin (Immucyst, Sanofi Pasteur), live H1N1 attenuated influenza A/PR8 (IAV, 100 HAU) (Charles River), a superantigen Enterotoxin (SEB, 0.4 μg/mL) (Bernhard Nocht Institute), and a Null control. 1 mL of whole blood was distributed into each of the prewarmed TruCulture tubes, inserted into a dry block incubator, and maintained at 37°C room air for 22 h. At the end of the incubation period, tubes were opened and a valve was inserted in order to separate the sedimented cells from the supernatant and to stop the stimulation reaction. Liquid supernatants were aliquoted (for paired oxylipin and cytokine analysis) and immediately frozen at 80°C until the time of use.

TruCulture whole-blood stimulations were performed in a standardized way as previously described^{4 (link),43 (link)}. Briefly, tubes were prepared in batch with the indicated stimulus, resuspended in a volume of 2 ml buffered medium, and maintained at −20 °C until time of use. Stimuli used in this study were LPS derived from E. coli O111:B4 (Invivogen), E. coli O111:B4 (Invivogen), C. albicans (Invivogen), vaccine-grade poly I:C (Invivogen), live Bacillus Calmette-Guerin (Immucyst, Sanofi Pasteur), live H1N1 attenuated influenza A/PR8 (IAV) (Charles River), SEB (Bernhard Nocht Institute), CD3 + CD28 (R&D Systems and Beckman Coulter), and cytokines TNF (Miltenyi Biotech), IL-1β (Peprotech) and IFNγ (Boehringer Ingelheim). One millilitre of whole blood was distributed into each of the prewarmed TruCulture tubes, inserted into a dry block incubator, and maintained at 37 °C room air for 22 h. At the end of the incubation period, tubes were opened, and a valve was inserted in order to separate the sedimented cells from the supernatant and to stop the stimulation reaction. Liquid supernatants were aliquoted and immediately frozen at −80 °C until the time of use.