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Then, 5 μL of IFN-γ catch reagent and 2.5 mL of warm (37°C) RPMI 1640 containing 10% human serum were added to the blood samples; the mixture was agitated at 90 rpm for 2 hours at 37°C (agitation is crucial to avoid IFN-γ capture by non-secreting cells).
The antibody-protein mixture was agitated at 1500 r.p.m. with Protein A/G agarose beads (Santa Cruz Biotechnology) for 1 h at 4 °C.
The mixture was agitated on a shaker for 5 h.
The mixture was agitated at 120 rpm at 30 °C until equilibrium was reached.
The mixture was agitated at room temperature (25 °C) for 12 h.
The reaction mixture was agitated at 100 °C for 4 h.
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Thereafter, the mixtures were agitated for 24 h.
The flasks were stoppered and the mixtures were agitated until equilibrium was attained.
The mixtures were agitated and incubated at room temperature in the dark.
The adsorbent dosage of 1.0 g/100 mL was used and the mixtures were agitated for 30 min.
The mixtures were agitated at approximately 200 rpm with a magnetic stirrer.
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