Application

This kit can be used for detection of low-density lipoprotein cholesterol (LDL-C) content in serum, plasma, cells, culture supernatant and tissue samples. 

Detection principle 

The coloured substance have a maximum absorption peak at 546 nm. Measure the OD value at 546 nm and the LDL-C content in the sample can be calculated.

Experimental instrument

Test tube, Micropipettor, Homogenizer, 37℃ water bath/gas bath, Microplate reader or Biochemical analyzer

Sample preparation

1. Serum (Plasma): Detect the sample directly. If the concentration is beyond the linear range, then dilute the sample with saline before detection.

2. Cell culture supernatant: Centrifuge at 1000 rpm for 10 min and then take the supernatant for test.

[Note]: It is generally recommended that the cell density should be more than 1×106/mL.

3. Tissue sample: Accurately weigh the tissue sample, add the homogenate medium at the ratio of Weight (g): Volume (mL) = 1: 9. Make the mechanical homogenization in ice water bath to prepare 10% homogenate. Centrifuge at 2500 rpm for 10 min, then take the supernatant for detection.

[Note]: (1) For high fat sample, the homogenate medium should be absolute alcohol.

(2) For non-high fat sample, the homogenate medium should be phosphate buffer (0.1 mol/L, pH 7.4) or normal saline.

4. Cell sample:

(1)  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 (0.1mol/L, pH7~7.4 phosphate buffer was recommended) 1~2 times, centrifuge at 1000 rpm for 10min, then discard the supernatant and keep the cell sediment.

(2)  Cell disruption: Add 0.2~0.3 mL of homogenate medium (0.1 mol/L, pH7~7.4 phosphate buffer or normal saline was recommended). Then treat the sample with sonication on ice (power: 300 W, 3~5 second/time, interval for 30 sec, repeat for 3~5 times) or grind with hand-operated. Take the prepared homogenate for detection without centrifugation. The cell can also be lysed with the cell lysate buffer (Triton X-100, 1~2%, lyse for 30~40 min), then take the prepared sample for detection without centrifugation. 

[Note]: It is generally recommended that the cell density should be more than 1×106/mL. The disrupted cell can be observed with microscope to check that whether the cell is broken completely.

Operation steps

1. Operate with 96 wells microplate.

2. Operate with Automatic biochemical analyzer.

a. Setting parameter

b. Operation steps

Performance index

1. The absorbance of the blank tube is ≤ 0.050 (Optical path is 0.5 cm).

2. Sensitivity: The difference of absorbance value △A is between 0.180~0.280 when testing 2.6 mmol/L samples.

3. Linear range: 0.02~12 mmol/L, r2 > 0.995.

4. Precision: the intra-assay CV ≤ 8%, the inter-assay CV≤ 10%.

5. Stability: This kit can be stored at 2~8℃ with shading light for 12 months and can be stable for 1 month since it is opened with the same storage condition.

Notes

1. This kit is for research use only.

2.  Instructions should be followed strictly, changes of operation may result in unreliable results.

3. The validity of kit is 12 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, then multiply by the dilution ratio when calculate the result.

6. Protect the reagent from contamination of glucose, cholesterol, etc.

7. The amount of reagent and sample can be increased and decreased according to the requirement of automatic biochemical analyzer.