Kimble kontes blue pellet pestles

Female C57bl/6J mice 8-9 weeks old (Jackson laboratories) were treated by I.P. injection with indicated compounds at the indicated times and doses in the text. Mice were euthanized by rapid asphyxiation with CO₂. Solid tissues were excised, rinsed in ice-cold PBS containing indomethacin (5.6 μgm/ml), and snap frozen in liquid nitrogen. Frozen samples were pulverized over liquid nitrogen. The powder transferred to an Eppendorf tube with 500 μl of cold PBS with indomethacin, and then homogenized using Kimble Kontes Pellet Pestle Cordless Motor (Fisher, K749540-0000) with Kimble Kontes blue pellet pestles (Fisher, K749521-1590) for ~30 s on ice. Then the suspension was sonicated in sonicator containing an ice water bath for 5 min using cycles of 20 seconds of sonication with 20 seconds of cooling, followed by centrifugation for 10 minutes at 12,000 rpm. The PGE2 level of the supernatant was measured using a PGE2 ELISA Kit (Enzo cat. #ADI-931-001) and normalized to protein concentration measured by BCA assay (Thermo Scientific, cat. #23225) and expressed as ng PGE2/mg protein. Each sample was assayed in triplicate.

8-9 Week old female C57bl/6J mice (Jackson laboratories) were treated by oral gavage with indicated compounds at the indicated times and doses in the text. Compounds were formulated as described in the pharmacokinetics studies. Mice were euthanized by rapid asphyxiation with CO₂. Solid tissues were excised, rinsed in ice-cold PBS only and snap frozen in liquid nitrogen. Frozen samples were pulverized over liquid nitrogen. The powder was transferred to an Eppendorf tube with 500 μl of cold lysis buffer (50 mM Tris-HCl, pH 7.5, 0.1 mM DTT, 0.1 mM EDTA) and then homogenized using Kimble Kontes Pellet Pestle Cordless Motor (Fisher, K749540-0000) with Kimble Kontes blue pellet pestles (Fisher, K749521-1590) for ~30s on ice. The suspension was centrifuged for 10 minutes at 12,000 rpm. The enzyme activity was measured by transfer of tritium from a tritiated PGE2 substrate to glutamate by coupling 15-PGDH to glutamate dehydrogenase with 1 mM NAD⁺, 5 mM NH₄Cl, 1mM α-keto-Glutarate (Sigma#75890) and 16 U of Bovine Liver Glutamic dehydrogenase (Sigma, cat. #G-2501) as described previously.^{17 (link)} The activity was normalized to protein concentration measured by BCA assay (Thermo Scientific, cat. #23225) and expressed as CPM/Hour/mg protein.