Centrifuge the cells (3000 rpm,
10 min) and then collect the supernatant
to remove cell debris. Add a mixture containing 20 μL of the
bead mixture, 20 μL of the sample solution, and 20 μL
of the assay antibody mixture incubated for a 96-well filter plate
and for 2 h at 500 rpm in the dark environment. Then, 20 μL
of PE-labeled Streptomyces-plant cyanate
(SA-PE) solution is put in the wells for 30 min (500 rpm) at room
temperature. Lastly, the corresponding fluorescence is measured by
flow cytometry and the level of intracellular cytokines in the sample
is obtained by analyzing the fluorescence intensity of the immune
complex; fluorescence data were collected using BD FACS Diva software
and intracellular cytokine levels were evaluated using FCAP Array
3.0 analysis software.31 (link)
10 min) and then collect the supernatant
to remove cell debris. Add a mixture containing 20 μL of the
bead mixture, 20 μL of the sample solution, and 20 μL
of the assay antibody mixture incubated for a 96-well filter plate
and for 2 h at 500 rpm in the dark environment. Then, 20 μL
of PE-labeled Streptomyces-plant cyanate
(SA-PE) solution is put in the wells for 30 min (500 rpm) at room
temperature. Lastly, the corresponding fluorescence is measured by
flow cytometry and the level of intracellular cytokines in the sample
is obtained by analyzing the fluorescence intensity of the immune
complex; fluorescence data were collected using BD FACS Diva software
and intracellular cytokine levels were evaluated using FCAP Array
3.0 analysis software.31 (link)
