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Classic activation of 3D4/31 porcine alveolar macrophages using phorbol 12‐myristate 13‐acetate and ionomycin
Author(s) -
Myers Jay L,
Tagliaferro Anthony R
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.908.4
Subject(s) - ionomycin , phorbol , incubation , chemistry , andrology , fetal bovine serum , alveolar macrophage , secretion , pulmonary alveolus , tumor necrosis factor alpha , microbiology and biotechnology , immunology , in vitro , cell culture , bovine respiratory disease , macrophage , biology , biochemistry , medicine , genetics , protein kinase c , enzyme
We hypothesize that dietary n‐6 fatty acids can convert alveolar macrophages to an alternative phenotype that promotes a Th2 immune response and respiratory allergy in humans. Our purpose was to establish an in vitro model using a transformed line of porcine alveolar macrophages (3D4/31). Cell populations of 3D4/31 cells were cultured in RPMI growth media (GM) supplemented with 10% fetal bovine serum (FBS) and incubated at physiological conditions (5% CO2 and 37 C). Three 24‐well plates were seeded at 100,000 cells/well and divided by serum conditions; 10% FBS or 10% porcine serum (PorS). After a 24‐hour incubation, wells were inoculated with phorbol 12‐myristate 13‐acetate (PMA; 0.01 mg/ml) and ionomycin (0.05 mg/ml). At various time points up to 12 hours, conditioned media was removed, centrifuged and stored at ‐80 C until ELISA analysis for TNF‐a and IL‐10. Total mean TNF‐a secretion was 22% higher when using PorS supplemented GM compared to FBS (n=18; p<0.05). Between 3 and 6 hours post‐activation, mean TNF‐a concentrations increased linearly and PorS supplemented GM was 29% greater than FBS (p<0.05) at 6 hours. The media was not found to contain measurable levels of IL‐10. 3D4/31 cells secrete TNF‐a and not IL‐10 when activated by PMA/ionomycin and are capable of providing a classically activated macrophage model to investigate potential influences of n‐6 fatty acids. UNH Experimental Station Project H495.