The cells were harvested in lysis buffer (Promega) and were assayed by using the dual luciferase system (Promega).
Transcription reactions contain buffer control, Poros-HE fractions and are assayed as in (B ).
Three venom amounts (diluted in 100 μL of ELISA buffer) were assayed: 16,000 ng, 2,000 ng, and 40 ng.
As proof of concept of the translatability of the fluorescent protocol to human research, a series of tests on consecutive dilutions of known cytokines in buffer were assayed.
Forty eight hours after transfection, cells were lysed in Tropix lysis buffer and lysate was assayed for luciferase activities.
These cells were lysed using a passive lysis buffer after 24 h, and lysates were assayed for luciferase activity using a luminometer TD-20/20 TD-20/20a) according to the manufacturer's instructions.
The cells were treated with lysis buffer (Promega) and luciferase activities were assayed by a luminometer (MicroLumat Plus LB96V; Berthold, Wilbad, Germany).
20S proteasome activities were determined similarly but using different reaction buffers: β1 and β2 activities were assayed in 25 mM HEPES, pH 7.5, 0.5 mM EDTA, 0.05% NP-40, 0.001% SDS.
To measure NF-kB activity, cells stimulated with LPS or PGN, were lysed in 30 µL of passive lysis buffer, and 10 µL of lysate were assayed for Firefly and Renilla activity according to the manufacturer's instructions.
Each male was recovered into lysis buffer and males scoring as mutant were assayed for a centrally located SNP on each chromosome (Wicks et al., 2001).
