IL-8 Biological Activity Assay

Summary

There are activating and chemotactic effects on neutrophils in IL-8, and the biological activity of IL-8, i.e., chemotactic activity on neutrophils, can be determined by detecting the migration distance of neutrophils.

Operation method

IL-8 biological activity assay

Materials and Instruments

IL-8 induction
Dextrose saline Agarose Lymphocyte layering solution Hank's solution Methanol Wright-Giemsa staining solution
Clean slides Perforator Centrifuge Incubator

Move

I. Induction of IL-8
1. Routinely isolate PBMC, wash and adjust the cell concentration to 5×10⁶/ml.
2. Inoculate cells into 24-well cell culture plates at 0.5 ml per well.
3. Add inducer LPS (20 ug/ml), 0.5 ml per well, incubate at 37 degrees Celsius, 5% _CO2 for 48 h.
4. Collect the cell culture supernatant by centrifugation, adjust it to be acidic (pH 4.0) with HCl solution, separate it by SephadxG-75 column, and collect the active component which is the crude IL-8 to be measured.
II. Bioactivity assay of IL-8
1. Preparation of agar plate; take 2% agarose which is dissolved and kept warm at about 50 degrees and equal volume of DMEM culture solution (2×concentrated) pre-warmed in water bath at 50 degrees, mix it, take 3 ml and add it to clean slide, spread it into a thin layer, solidify it at room temperature and put it at 4 degrees Celsius, solidify it for further 30~60 minutes, and red punch the holes.
2. Neutrophil preparation: routine isolation of human peripheral blood neutrophils.
3. Sample addition: take 10 ul neutrophil suspension and add it to the center well of each group of agar plate, take 10 ul of the sample to be examined and the positive control and add it to the sample well, take 10 ul of DMEM culture solution and add it to the control well; put the slides into wet box and incubate them at 37℃ for 2 hours.
4. Staining: fixation in methanol solution for 30 minutes, staining of the slides with Rachel's stain and drying.
5. Detection: Microscopic measurements of the distance traveled by the cells from the central pore to the sample pore (chemotaxis travel distance) and to the positive control pore (spontaneous travel distance), and chemotaxis activity is expressed as chemotaxis index.
6. Convergence index = convergent travel distance/spontaneous travel distance

Caveat

To confirm the specific chemotactic effect of IL-8 in the samples to be examined, it is desirable to compare the chemotactic results of the anti-IL-8 antibody acting on the samples to be tested at the same time.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/