Mouse scf quantikine elisa kit

To harvest bone marrow interstitial fluid, long bones were flushed into 1.5 ml Eppendorf tubes using compressed air and immediately weighed and dissociated in 15-fold DPBS by pipetting and vortexing. The mixture was then centrifuged at 100 g for 5 min, and the supernatant was transferred to new tubes, followed by another centrifugation at 9200 g for 5 min to remove the remaining debris. To harvest serum, blood was collected and allowed to clot at room temperature for 1 h. The clotted blood was then centrifuged at 150 g for 10 min and the serum was transferred to new tubes. The serum was diluted 5-fold for the assay. ELISA for SCF and TPO was conducted with Mouse SCF Quantikine ELISA Kit (R&D, MCK00) and Mouse Thrombopoietin Quantikine ELISA Kit (R&D, MTP00).

Lysates of whole fetal spleen or liver tissues were prepared using the Qproteome Mammalian Protein Prep Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. Hematopoietic cells (CD45+/CD45+Ter119+/Ter119+), endothelial cells (CD45−Ter119−CD31+LYVE-1−), unclassified cells (CD45−Ter119−CD31−LYVE-1−) and CD51+ cells among unclassified cells (CD45−Ter119−CD31−LYVE-1−CD51+) were sorted. Lysates were prepared using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Rockford, IL) and a protease inhibitor from the Qproteome Mammalian Protein Prep Kit (Qiagen). Samples were centrifuged at 12,000 rpm for 10 minutes at 4°C. Supernatants containing soluble protein were collected and protein concentration was quantitated using the Bradford reagent (Bio Rad, Hercules, CA) according to the manufacturer's instruction. The optical density (O.D.) at 540 nm was measured using a Thermo Multiskan FC plate reader (Thermo Fisher Scientific). SCF, IGF-1, IL-3 and EPO ELISA assays were conducted using a mouse SCF Quantikine ELISA kit (R&D Systems, Minneapolis, MN), a mouse IGF-1 Quantikine ELISA kit (R&D Systems), a mouse IL-3 Quantikine ELISA kit (R&D Systems) and a mouse EPO Quantikine ELISA kit (R&D Systems), according to the manufacturer's instructions. O.D.s at 450 nm and 540 nm were measured using a Thermo Multiskan FC plate reader.