Application

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

Detection principle

The generated red purple pigment have a maximum absorption peak at 546 nm. Measure the OD value at 546 nm and the HDL-C content in the sample can be calculated.

Experimental instrument

Test tube, Micropipettor, Vortex mixer, Water bath, Spectrophotometry (546 nm)

Sample pretreatment

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 1×106 /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 1×106/ml. The disrupted cell can be observed with microscope that whether the cell is broken completely.

Operation steps

Performance index

1. The absorbance of blank tube is ≤ 0.010 (optical path = 0.5 cm).

2. Linear range: 0.065~3.8 mmol/L, r2 > 0.995.

3. Sensitivity: The absorbance value of △A is between 0.087 ~ 0.153 when testing 1.3 mmol/L samples.

4. Accuracy: Relative deviation ≤ 10%.

5. Precision: intra-CV≤ 3%, inter-CV≤ 5%.

6. Stability: The validity of kit is 12 months when stored at 2℃~8℃ in the dark. It is stable for 1 months when stored at 2℃~8℃ in the dark after opening.

Notes

1. This product is for scientific research use only, not for clinical diagnosis.

2. The validity of kit is 12 months.

3. Do not use components from different batches of kit.

4. 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.

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

6. The amount of reagent and sample can be increased and decreased proportionately according to the volume of cuvette.