Description

Description: Application This kit applies the COD-PAP method and it can be used for in vitro determination of total cholesterol (T-CHO) content in serum, plasma, cells, culture supernatant and other samples. Detection significance Cholesterol is a kind of sterol and lipid in cell membrane. Most of cholesterol in blood exists in the form of cholesterol ester. Lecithin-cholesterol acyltransferase in human plasma is an enzyme that catalyzes the formation of cholesterol ester. Cholesterol synthesized or deposited in peripheral cells returns to the liver through the reverse cholesterol transport system for reusing or regaining bile acids. Detection principle The color depth of the generated quinones is directly proportional to the cholesterol content. The absorbance values of the standard tube and the sample tube are measured respectively, and the cholesterol content in the sample can be calculated. If the total cholesterol (T-CHO) content is calculated by protein concentration, the protein concentration of the sample needs to be determined separately (E-BC-K318, E-BC-K168, E-BC-K165). The preparation of sample 1. Serum (Plasma): Detect the sample directly. If the concentration is beyond the linear range, then dilute the sample with normal saline before detection. 2. Culture supernatant sample: Collect the culture medium, centrifuge at 1000 rpm for 10 min, and take the supernatant for detection. [Note]: It is generally recommended that the cell density should be more than 1x106 /mL. 3. Tissue sample: Accurately weigh the tissue weight, add 9 times the volume of homogenate media according to the ratio of Weight (g): Volume (mL) =1:9. Mechanical homogenate the sample in ice water bath. Centrifuge at 2500 rpm for 10 min, then take the supernatant for detection. [Note]: (1) If the tissue sample is not a high-fat sample, the homogenate media should be phosphate buffer (0.1 mol/L, pH 7.4) or normal saline. (2) If the tissue sample is high-fat sample or partly high lipid sample, the homogenate media should be absolute alcohol. 4. Cell sample: Cell collection: Take the prepared cell suspension and centrifuge at 1000 rpm for 10 min. Discard the supernatant and keep the cell sediment. Wash the sediment with iso-osmia buffer (0.1 mol/L, pH7~7.4 phosphate buffer was recommended) 1~2 times, centrifuge at 1000 rpm for 10 min. Discard the supernatant and keep the cell sediment. Cell disruption: Add 0.2~0.3 mL of homogenate media (0.1 mol/L, pH7~7.4 phosphate buffer or normal saline was recommended). Sonicate in ice water bath (power: 300 W, 3~5 second/time, interval for 30 sec, repeat for 3~5 times) or grind with hand-operated. The prepared homogenate kept for detection without centrifugation. The cell can also be lysed with the cell lysate buffer (Triton X-100, 1~2%, 30~40 min), then take the prepared lysate for detection directly without centrifugation. [Note]: It is generally recommended that the cell density should be more than 1x106/ml. The disrupted cell can be observed with microscope that whether the cell is broken completely. Operation steps It is recommended to take 2~3 samples which expected large difference to do pre-experiment before formal experiment. 1. Blank tube: add 2.5 µL of double distilled water into the well. Standard tube: add 2.5 µL of Standard solution into the well. Sample tube: add 2.5 µL of sample into the well. 2. Add 250 µL of working solution to each well. 3. Mix thoroughly, incubate at 37°C for 10 min, measure absorbance value A at 510 nm with microplate reader. Note: It can be refer to the following operating table. Technical parameters 1. The sensitivity of the kit is 0.29 mmol/L. 2. The intra-assay CV is 3.1% and the inter-assay CV is 8.3%. 3. The recovery of the kit is 103%. 4. The linear range of the kit is 0.29-25.85 mmol/L. Notes 1. This product is for scientific research use only. 2. Instructions should be followed strictly, changes of operation may result in unreliable results. 3. The validity of the kit is 6 months. 4. Do not use components from different batches of kit. 5. If the sample content is beyond the maximum limit, please dilute the sample with normal saline before detection, and multiply the result by the dilution ratio. 6. Protect the reagent from contamination of glucose, cholesterol, etc. 7. Prevent the formulation of bubbles when the reagents is added into the microplate.

Additional Text

2-8°C