Sample Collection And Storage
Serum - 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 - The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissues were rinsed in ice-cold PBS(0.02mol/L,pH 7.0-7.2) to remove excess blood thoroughly and weighed before homogenization. Minced the tissues to small pieces and homogenized them in 5-10 mL of PBS with a glass homogenizer on ice(Micro Tissue Grinders woks, too). The resulting suspension was sonicated with an ultrasonic cell disrupter or subjected to two freeze-thaw cycles to further break the cell membranes. After that, the homogenates were centrifugated for 5 minutes at 5000×g. Remove the supernate and assay immediately or aliquot and store at ≤-20℃.
Cell Lysates - Cells must be lysed before assaying according to the following directions.
1. Adherent cells should be detached with trypsin and then collected by centrifugation (suspension cells can be collected by centrifugation directly).
2. Wash cells three times in cold PBS.
3. Resuspend cells in PBS (1×) and the cells was subject to ultrasonication for 4 times (or Freeze cells at≤-20℃. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle for 3 times.)
4. Centrifuge at 1500×g for 10 minutes at 2 - 8℃ to remove cellular debris.
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℃. Avoid repeated freeze/thaw cycles.