SAMPLE COLLECTION AND STORAGE

Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissues were rinsed in ice-cold PBS(0.01mol/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^oC.

 组织匀浆：

1） 取适量组织块，于预冷PBS（0.01mol/L, pH 7.0-7.2）中清洗去除血液，称重后备用（组织块较大需先剪碎后再匀浆）；

2） 可同时选用多种匀浆方法达到较好的破碎效果：首先将组织块移入玻璃匀浆器，加入5-10mL预冷PBS进行充分研磨，该过程需在冰上进行（有条件实验室可选用机器匀浆）；得到的匀浆液可再利用超声破碎或反复冻融进一步处理（超声破碎过程中注意冰浴降温；反复冻融法可重复2次）。

3） 将制备好的匀浆液于5000×g离心5分钟，留取上清即可检测。