B3056) dual PKA/PKC inhibitor (Ki = 3.0��M and 6.0��M, resp.), adenylyl cyclase http://www.selleckchem.com/products/Nilotinib.html inhibitor SQ 22,536 (ref.S153), 6-Isopropoxy-9-oxoxanthene-2-carboxylic acid (AH6809) (ref.A1221), selective murine EP2 antagonist [18], and 2-acetylhydrazide 10(11H)-carboxylic acid, 8-chloro-dibenz[b,f][1,4]oxazepine-10(11H)-carboxylic acid (SC19220) (ref.S3065), preferential EP1 receptor antagonist from Sigma Chemical Co. (St Louis, MO); dibutyryl cyclic AMP (ref.D0260), forskolin (ref.F6886), Z-VAD-fmk (ref.V116), dexamethasone 21-phosphate disodium salt (ref.D1159), and aminoguanidine hydrochloride, a selective iNOS inhibitor (ref.396494) from Sigma-Aldrich (St. Louis, MO); biotinylated anti-mouse IgG antibody (ref. SC-2040) from Santa Cruz Biotechnology (Santa Cruz, CA, USA.
); rolipram from Sanofi (Montpellier, France); 4-amino-5-methylamino-2,7-difluorescein diacetate (DAF-FM) (ref.”type”:”entrez-nucleotide”,”attrs”:”text”:”D23844″,”term_id”:”427709″D23844) from Invitrogen (S?o Paulo, Brazil) [19]; liquid diaminobenzidine (DAB+) (ref. K3467) solution from Dako Cytomation (Dako Denmark A/S, Glostrup, Denmark).2.3. Bone-Marrow Culture Liquid bone-marrow culture conditions were routinely used because they allow addition of agonists and inhibitors at different times, which cannot be done in clonal culture systems, where proper mixing and diffusion of substances added after plating is restricted by the semisolid media. The previously reported effects of PGE2 and cAMP-elevating agents [12] in semisolid culture are nevertheless consistent with their suppressive effects in liquid culture as detailed here (see Results Section 3.
1). Eosinophilopoiesis in liquid culture was strictly dependent on IL-5 [7], and culture conditions were adequate for demonstrating both enhancing and suppressive effects [12]. BM cells were collected from both femurs of naive mice, washed, counted in a haemocytometer, seeded at 106 in 1mL of RPMI 1640 medium, 10% FCS, and rmIL-5 (1ng/mL; optimal dose, as previously defined [7]) in 24-well clusters, and incubated at 37��C, 5% CO2/95% air for 7 days. Where indicated, cultures received PGE2, isoproterenol, or one of the cAMP-elevating agents (dibutyryl-cAMP, rolipram, cholera toxin, or forskolin) known to duplicate the effects of PGE2 in semisolid culture [12]. Unless otherwise indicated, each agonist was added only once, immediately after IL-5, at the beginning of the culture period.
Carfilzomib In selected experiments, PGE2 was added at various times after initiation of the culture [9]. Where indicated, cultures also received EP inhibitors (AH6809 or SC19220), iNOS inhibitor (aminoguanidine) [8], adenylyl cyclase inhibitor (SQ22536), or protein kinase A and C inhibitors (H89 or Bis) or caspase inhibitor (zVAD-fmk) [20]. In this case, inhibitors were added before both IL-5 and the agonists they were expected to antagonize.