SAMPLE COLLECTION AND STORAGESSerum

SAMPLE COLLECTION AND STORAGESSerum - Use a serum separator tube and allow samples to clot for two hours at room temperature or overnight at 4℃ before centrifugation for 20 minutes at approximately 1000×g. Assay freshly prepared serum immediately or store samples in aliquot at -20℃ or -80℃ for later use. Avoid repeated freeze/thaw cycles.Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2-8℃ within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquot at -20℃ or -80℃ for later use. Avoid repeated freeze/thaw cycles.Tissue homogenates - For general information, hemolysis blood may affect the result, so you should rinse the tissues with ice-cold PBS (0.01M, pH=7.4) to remove excess blood thoroughly. Tissue pieces should be weighed and then minced to small pieces which will be homogenized in PBS (the volume depends on the weight of the tissue. 9mL PBS would be appropriate to 1 gram tissue pieces. Some protease inhibitor is recommended to add into the PBS.) with a glass homogenizer on ice. To further break the cells, you can sonicate the suspension with an ultrasonic cell disrupter or subject it to freeze-thaw cycles. The homogenates are then centrifugated for 5minutes at 5000×g to get the supernate.Cell culture supernates and other biological fluids - Centrifuge samples for 20 minutes at 1000×g. Remove particulates and assay immediately or store samples in aliquot at -20℃ or -80℃ for later use. Avoid repeated freeze/thaw cycles.