[ MATERIALS REQUIRED BUT NOT SUPPLIED ]
1. Microplate reader with 450 ± 10nm filter.
2. Precision single or multi-channel pipettes and disposable tips.
3. Eppendorf Tubes for diluting samples.
4. Deionized or distilled water.
5. Absorbent paper for blotting the microtiter plate.
6. Container for Wash Solution
[ STORAGE OF THE KITS ]
1. For unopened kit: All the reagents should be kept according to the labels on vials. The Standard, Detection
Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20oC upon receipt while the
others should be at 4 oC.
2. For opened kit: When the kit is opened, the remaining reagents still need to be stored according to the above
storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and
reseal along entire edge of zip-seal.
Note:
It is highly recommended to use the remaining reagents within 1 month provided this is within the expiration date
of the kit.
[ SAMPLE COLLECTION AND STORAGE ]
Serum - Use a serum separator tube and allow samples to clot for two hours at room temperature or overnight at
4oC before centrifugation for 20 minutes at approximay 1000×g. Assay freshly prepared serum
immediay or store samples in aliquot at -20oC or -80oC 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 - 8oC within 30 minutes of collection. Remove plasma and assay immediay or store samples
in aliquot at -20oC or -80oC 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 immediay or aliquot and store at ≤-20oC.
Cell culture supernates and other biological fluids - Centrifuge samples for 20 minutes at 1000×g. Remove
particulates and assay immediay or store samples in aliquot at -20oC or -80oC for later use. Avoid repeated
freeze/thaw cycles.
Note:
1. Samples to be used within 5 days may be stored at 4oC, otherwise samples must be stored at -20oC （≤1
month） or -80oC （≤2 months） to avoid loss of bioactivity and contamination.
2. Sample hemolysis will influence the result, so hemolytic specimen can not be detected.
3. When performing the assay, bring samples to room temperature.