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 aliquots at -20℃ or -80℃ for later use. Avoid repeated freeze/thaw cycles.
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 aliquots at -20℃ or -80℃ for later use. Avoid repeated freeze/thaw cycles.
The preparation of tissue homogenates will vary depending on tissue type. In general, tissues should be rinsed thoroughly in ice-cold PBS (0.02mol/L,pH 7.0-7.2) to remove excess blood and weighed before homogenization. Mince the tissues to small pieces and homogenize them in 5-10 mL of PBS with a glass homogenizer on ice (Micro Tissue Grinders work as well). The resulting suspension should be sonicated with an ultrasonic cell disrupter or subjected to two freeze-thaw cycles to further break the cell membranes. After that, the homogenates should be centrifugated for 5 minutes at 5000×g. Remove the supernatant and assay immediately or aliquot and store at ≤-20 ℃.
Cells must be lysed before assaying according to the following directions:
- Adherent cells should be detached with trypsin and then collected by centrifugation (suspension cells can be collected by centrifugation directly).
- Wash cells three times in cold PBS.
- Resuspend cells in 1X PBS and subject to ultrasonication 4 times (or Freeze cells at≤-20℃. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle 3 times.)
- 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.