Briefly, samples were stimulated in the indicated conditions for 24 hours then washed without removing the CD3/CD28 beads and resuspended in PBS with 2 µM DCFDA.
After addition of FITC-conjugated anti-human CD42b and PE-conjugated anti-human CD14, samples were stimulated with ADP (1 3 μM) and incubated for 20 min at 25 °C then fixed using paraformaldyde (4%, Sigma Aldrich, Poole, UK) and stored at 4 °C prior analysis.
Diluted 1 10 whole-blood samples were stimulated with 500 pg/ml LPS, at 37°C, for 4 hours.
All samples were stimulated within 5 h after blood collection.
All aliquots of the blood samples were stimulated within four hours of sampling.
Total RNA was purified from blood samples that were stimulated for 4 hours with Gardiquimod™ (TLR7), ssRNA (TLR8) and CL075 (TLR7/8) and mRNA levels measured by real time PCR.
For detection of intracellular IL-6, samples of whole blood were stimulated for 6 hours.
(B) samples from gecko (T. mauritanica); lizard (A. Carolinensis); snake (T. elegans; mouse NIH3T3 were stimulated for 10 min prior to lysis (lane1) or were stimulated one hour in presence of MEK inhibitor PD184352 (lane2) to block ERK phosphorylation by MEK Kinase.
[37] Cells were stimulated for 48 hours and cell lysates were collected in 5XSDS-PAGE sample buffer (Sigma-Aldrich, St . Louis MO).
The cells were stimulated for 24 hours.
