IL-1 biological activity assay

Summary

Interleukin-1 (IL-1) is a cytokine mainly synthesized and secreted by activated monocyte-macrophage cells, and has various biological functions such as activation of lymphocytes, synergistic stimulation of thymocyte proliferation, participation in antibody production, and pro-inflammatory response.IL-1 is composed of IL-1α and IL-1β, which bind to the same kind of receptor and exhibit the same biological activity.

Operation method

Mouse thymocyte proliferation assay

Principle

IL-1 stimulated mouse thymocyte proliferation when co-cultured with mouse thymocytes. The biological activity of IL-1 was further presumed by determining the amount of mouse thymocyte proliferation by the 3H-TdR doping method or the dye uptake method. Detection of the biological activity of IL-1 provides insight into the functions of monocytes and macrophages.

Materials and Instruments

Mouse Thymus Cells
FCS-RPMI-1640 culture medium LPS Sword bean protein A 3H-TdR

Move

1. Induction of IL-1 production by mouse macrophages
(1) Mouse peritoneal macrophages were routinely collected, and the cells were suspended in 10% FCS-IMDM culture solution at ^2×106/ml and inoculated in 24-well culture plates at 0.5 ml/well.
(2) Add 20 ug/ml LPS 0.15 ml per well and incubate at 37 degrees, 5% CO2 for 36~48 hours.(3) At this time, the culture supernatant contains high levels of IL-1 secreted by macrophages, collect the culture supernatant, centrifuge at 12000 r/min for 15 minutes, transfer the supernatant into a new 1.5 ml test tube, and freeze at -20℃.
2. Determination of biological activity of IL-1
(1) Preparation of thymocytes: cervical dislocation mice, aseptically take the thymus to a flat dish, add 5 ml 10% FCS-IMDM culture medium, 200 mesh stainless steel mesh to make a single cell suspension; centrifugation at 1500 r/min for 10 minutes, and then 5% Hank's solution to wash the cells for two times, then resuspended in 10% FCS-IMDM culture medium, and adjust the cell concentration to 1. ^0×107/ml.(2) Addition of samples: Take thymocytes and add them to 96-well cell culture plate, 0.1 ml/well, then add IL-1 samples to be tested or IL-1 standard, 50 ul/well, and then add 50 ul ConA (final concentration 0.625 ug/ml) to the experimental wells, and at the same time, set up the culture medium control wells (0.1 ml of cells + 0.1 ml of culture medium), the ConA control wells ( 0.1 ml of cells + 50 ul culture medium + 50 ul ConA), all set up 3 duplicate wells. 0.1 ml cells + 50 ul culture medium + 50 ul ConA), all set up 3 duplicate wells. 37 degrees, 5% _CO2 incubation 36~60 hours.(3) ^3H doping: 1. ^85×1010 uBq/20 ul (0.5 uCi/20 ul) ^3H-TdR was added to each well and incubation was continued for 8 hours.(4) Measurement: Cells were collected on glass fiber filter paper with a porous cell collector and ^3H-TdR was measured by β-liquid scintigraphy.(5) The amount of adulteration (cpm) was plotted on semi-logarithmic graph paper using the net cpm value (experimental wells - ConA control wells cpm value) as the vertical coordinate and the corresponding different dilution concentrations of IL-1 standard as the horizontal coordinate, and then the content of IL-1 in the samples to be tested was found out from the standard curve.

Caveat

1. When aspirating LPS-stimulated mouse macrophage culture supernatants, they must be centrifuged to remove cells and cell fragmentation components.

2. ConA should be selected as a sub-dose for lymphocyte transformation assays, i.e., an amount that can activate thymocytes without inducing significant proliferation; if ConA is in excess, it does not effectively respond to the stimulatory activity of IL-1.

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