Leukocyte movement inhibition assay
Leukocyte movement inhibition assay
Sensitized T-lymphocytes react with the corresponding antigen, causing metabolic activation and the release of a variety of biologically active substances. Among them are factors that inhibit the movement of monocytes and macrophages (MIF) and factors that inhibit the movement of polymorphonuclear leukocytes (LIF), which are not found in normal lymphocytes.
Operation method
Leukocyte movement inhibition assay
Principle
The presence of leukocyte movement inhibitory factor is examined by its biological activity to inhibit the movement of leukocytes. Depending on the way of observing the movement of leukocytes, there are three different methods of movement inhibition test, i.e., capillary method, agarose perforation method, and agarose microtitre method, and the capillary method is introduced here.
Materials and Instruments
Leukocytes Move 1. Lymphocyte Extraction. For more product details, please visit Aladdin Scientific website.
Cell Culture Solution Hanks Liquid Lymphocyte Extracts
Capillary Glass Tubes Cover Sheets Centrifuge Test Tubes Dropper Tubes
2. Use sterile tweezers to clip the capillary tube, inserted into the cell suspension, so that the cell suspension through the capillary phenomenon into the capillary tube, in the upper end of the 7~8mm, the capillary tube will be removed, loaded into each capillary tube, so that the height of the cell suspension seeks to relatively equal.
3. Place the empty end of the capillary tube, flame-sealed end down, in a test tube and centrifuge horizontally at 1000 rpm for 10 minutes.
4. Remove the capillary and see that the cells have been pressed into a column 4-6 mm high, with a whitish surface layer of predominantly leukocytes and a reddish lower layer of a mixture of red and leukocytes. Use a small grinding wheel to scratch the capillary wall at the cell-liquid interface, and be careful to break it.
5. Dip the capillary tube overflowing with cells into a little petroleum jelly, place it in a flat-bottomed concave glass and fix it, put 2~8 capillaries in each concave, and then add the culture medium dropwise, divide the capillaries into two groups, 3~4 in each group, one group is the experimental group, and the other group is the control group.
6. The experimental group was incubated with 199 culture plus appropriate amount of antigen as culture medium, while the control group was incubated with 199 liquid without antigen.
7. The concave hole was closed with a coverslip, do not leave air bubbles, the concave slide was placed in an aluminum box lined with wet cloth gauze, and incubated in a 37℃ incubator for 18~22hr.
8. Concave slides were removed, and the length and diameter of the sector in which each capillary leukocyte moved were determined by dissecting microscope. The results of the test were expressed as the movement index MI:
9. Normal values range from 80 to 120%; below 80% is considered inhibition, i.e., positive for mobile inhibition, and below 120% is considered stimulation, which may be due to a low antigen concentration.