Optimization of assay conditions for leukotriene B4 synthesis by neutrophils or platelets isolated from peripheral blood of monogastric animals

Dietary n-3 fatty acids decrease the leukotriene B4 response ex vivo and the bovine serum albumin-induced footpad swelling index in New Hampshire hens

“…Aliquots of 5 × 10 6 cells were transferred to test tubes and HBSS containing 0.8 mM CaCl 2 was added to achieve a final volume of 475 µL. Cells were preincubated at 37ºC for 10 min, and then stimulated with 25 µL calcium ionophore A23187 (final concentration 20 µM) or treated with 25 µL 0.2% DMSO (control) and incubated for 10 min at 37ºC as described by Jha et al (2005). Supernatants were stored at -70°C until subsequent LTB 4 and LTB 5 analyses were performed.…”

“…Leukotriene B 4 and LTB 5 were extracted, separated, and quantified using the methods described by Terano et al (1984) and updated by Jha et al (2005) for monogastric species including chickens. Standard calibration curves for LTB 4 and LTB 5 were made by adding 100 ng of prostaglandin B 3 (PGB 3 ) as an internal standard to samples containing 6.25, 12.5, 25, 50, or 100 ng of both LTB 4 and LTB 5 .…”

Dietary (n-3) fatty acids alter plasma fatty acids and leukotriene B synthesis by stimulated neutrophils from healthy geriatric Beagles

“…Neutrophils were isolated and purified as previously described [16] from blood collected at 36 weeks for the experimental dogs and at 4 weeks for the healthy Beagles used in the teaching program. Aliquots of 1 x 10 6 neutrophils were transferred to test tubes and the volume adjusted to 495 µL with Hanks balanced salt solution containing 0.8 mmol/L CaCl2.…”

“…Neutrophils were then stimulated with 5µL of calcium ionophore A23187 (Sigma Chemical Co., St. Louis, MO, USA) in 0.2% dimethyl sulfoxide (Syntex, West Des Moines, IA, USA) such that the final concentration of A23187 was 10 mmol/L, while unstimulated neutrophils received 5 µL of 0.2% dimethyl sulfoxide without calcium ionophore. All tubes were incubated for 5 min at 37'C as previously described [16]. Supernatants were stored at -70'C until subsequent LTB4 and LTB5 measurements were made.…”

“…Supernatants were stored at -70'C until subsequent LTB4 and LTB5 measurements were made. LTB4 and LTB5 were extracted, separated, and quantified as previously described [16]. Standard calibration curves for LTB4 and LTB5 were made by adding 100 ng of PGB3 as an internal standard to samples containing 0.47-40 ng of LTB4 and LTB5 (Sigma).…”