B Detection of cell-secreted antibodies
B Detection of cell-secreted antibodies
The following is an example of mitogen-induced antibody production by B cells using American continental protein. This method has been adapted for use with a variety of other stimulatory factors and lymphocytes Author: J.E. Colligan et al, Translator: Xuitao Cao et al, This experiment is from the "Comprehensive Guide to Immunology".
Operation method
B. Detection of cellular discrete antibodies Move Basic Programs Materials Peripheral blood single nucleated cells (PBMC; Module 8.1) RPMI- 5 and RPMI-IO complete media P W M solution 1 . Count PBMC (Appendix 3A) and adjust the concentration to 4 X 107 cells/2mL with RPMI-5 complete medium. Centrifuge at 400 g for IOmin at room temperature and discard the supernatant. Add RPMI-5 complete medium and repeat the wash at least 4 times. 2 . Recount the PBMC and adjust the concentration with RPMI-IO complete medium to 5X IO 5 cells/ml. Add 0.2 ml of cell suspension (IX IO 5 cells per well) to a 96-well plate. Replicate 2 wells for each experimental group. Avoid using wells on the periphery of the plate. For longer cell cultures, the peripheral wells should be filled with water to minimize evaporation of liquid from the experimental wells. 3 . Add 20ul PWM (final dilution 1 : 1 0 0 ) to induce PBM C activation. The background control group was not stimulated. The cells were incubated at 37°C in a 5% C02 cell culture incubator (7d for ELISPOT assay and IOd for ELISA). 4 . Collect cells for subsequent reverse vacuolar assay (Section 7.13), ELISPOT assay (Section 8.11) or culture supernatant for ELISA (Section 8.12). For more product details, please visit Aladdin Scientific website.
9 6-well flat-bottomed culture plate (Costar)