According to manufacturer's instructions, 1U of DNase and 1 µl of DNase 10× Reaction Buffer were added in 8 µl of RNA sample and incubated 30 minutes at 37°C.
Subsequently, an optimal concentration of fluorochrome-conjugated antibodies (between 0.06 0.5 µg/1×106 cells in 50 µl of FACS buffer) were added in various combinations to allow for dual or triple staining experiments and plates were incubated for 30 minutes on ice.
Serum samples, diluted 1 50 in incubation buffer, were added in duplicate (100 μl/well) and incubated for two hours at room temperature.
50 μL of dye-labeled CPMV and TMV diluted in 0.1 M potassium phosphate buffer were added in triplicate to a black 384-well plate at concentrations of 2.5 μM sCy5, 50 nM CPMV, or 5 nM TMV.
Five μL of the sample and 15 μL of lysing buffer were added in duplicates into each well, together with 180 μL p-nitrophenyl phosphate (pNP) solution (Sigma Aldrich) and incubated for 30 minutes at 37°C.
The next day, non-specific binding sites of the plate were blocked with 5 % FCS/PBS (100 μl/well, fetal calf serum, PAA laboratories, Austria) for 1 h at RT. Subsequently, serially diluted serum samples (1 100, 1 500, 1 1000, 1 2000, 1 4000 in blocking buffer) were added in triplicate wells (50 μl/well) and incubated at RT for 2��h.
In order to reduce the signal reflection, output buffer is added in this design.
The reaction was allowed to react for 15 min before 15 mg (0.02 mmol) of 9, dissolved in 2 mL of 0.05 M phosphate buffer, was added in one portion to the reaction mixture.
Subsequently 0.5 µg of biotin labeled DBS diluted in 50 µl 1X GMS buffer was added in the well and incubated at 4°C for 1 hour.
The buffer was added in 1 ml per 1 g of cells ratio to 1.5 ml Eppendorf tubes, which we vigorously shook five times for one minute in a Disruptor Genie (Scientific Industries Inc ,Bohemia, NY, U.S.A).
