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Collection and treatment of common samples for metabolism assay detection

2018-09-18Author:adminpraise:1

(This is for your reference only.)

1. Serum

Collect fresh blood and stand at 25℃ for 30 min to clot the blood. Then centrifuge at 2000 g for 15 min at 4℃. Take the upper light yellow clarified liquid layer which is the serum and preserve it on ice for detection. If it can’t be tested on the same day, it can be stored at -80℃ for a month.

2. Plasma
Collect fresh anticoagulant blood (Heparin is used as anticoagulant and concentration of heparin is 10-12.5 IU/mL blood), centrifuge at 700-1000 g for 10 min at 4℃. Take the upper light yellow clarified liquid layer which is the plasma (don't take white blood cells and platelets in the middle layer) and preserve it on ice for detection. If it can’t be tested on the day, it can be stored at -80℃ for a month.

3. Urine

Collect fresh urine and centrifuge at 10000 g for 15 min at 4℃. Take the supernatant and preserve it on ice for detection. If it can’t be tested on the same day, it can be stored at -80℃ for a month.


4. Saliva

Gargling with clear water, after 30 min saliva was collected and centrifuged at 10000 g at 4℃ for 5 min. Then take the supernatant for detection. If not detected on the same day, the saliva can be stored at-80℃ for a month.


5. 10% Tissue homogenate

Take 0.02-1g fresh tissue to wash with homogenization medium at 2-8℃ to remove blood cells. Absorb the water with filter paper and weigh. Homogenize at the ratio of the volume of homogenized medium (2-8℃) (mL): the weight of the tissue (g) =9:1, then centrifuge the tissue homogenate for 10 min at 10000 g at 4℃. Take the supernatant to preserve it on ice for detection. Meanwhile, determine the protein concentration of supernatant (E-BC-K318-M, E-BC-K168-M, E-BC-K165-M). If not detected on the same day, the tissue sample (without homogenization) can be stored at -80℃ for a month.


6. Cells
Suspension cells: centrifuge at 1000-2000 g for 10 min at 4℃ to collect cells and resuspend the cells with 2-5 mL 2-8℃ PBS (0.01 M, pH 7.4), centrifuge at 1000-2000 g for 10 min at 4℃ and discard supernatant. Add homogenized medium into the sediment according to the ratio of cells number (106): homogenized medium (μL) =1: 200-400. Mechanically homogenize the cells to break the cells fully (There is no obvious sediment, which can be observed with microscope). Then centrifuge at 10000 g for 10 min at 4℃. Take the supernatant and preserve it on ice for detection. If it can’t be tested on the day, the cell (without homogenization) can be stored at -80℃ for a month.
Adherent cells: discard the culture medium and wash the cells with PBS (0.01 M, pH 7.4) for once. Collect the cells with a cell scraper (it can't be treated with trypsin or EDTA) and then add 2-5 mL PBS and collect the cell suspension. Then see the operation steps of suspension cells.

Note:
1. Homogenized medium

Please refer to the instructions of kits.

2. Homogenized method:
(1) Manual homogenization: pour the sample (including homogenized medium) into the glass homogenization tube, hold the homogenization tube in the left hand, insert the lower end into the container containing ice-water mixture, insert the tamping rod vertically into the homogenization tube in the right hand, and rotate and grind up and down for dozens of times (6-8 minutes) to homogenize the tissue.
(2) Mechanical homogenate: put the weighed tissue into EP tube, add homogenized medium, and grind with tissue homogenate machine at 60 Hz and 90 s under the condition of ice-water bath to make tissue homogenate. The homogenate time of skin, muscle tissue and plant tissue can be appropriately prolonged.
(3) Ultrasonication: the ultrasonic generator was used to treat the cells for 30 s with the amplitude of 14 μm in the ice bath. Or treat the cells with ultrasonic cell disruptor (200 W, 2 s/time, interval for 3 s, the total time is 5 min).
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