2822
A. Baxter et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2817–2822
addition of 25 lL Microscint-40 on a Packard topcount
microplate scintillation counter.
References and notes
13. Peripheral blood mononuclear cells (PBMCs) were pre-
pared from human whole blood using LymphoPrep
separation, counted using a haemocytometer and diluted
to 2.6 ꢀ 106/mL in cell medium {RPMI 1640 supplemented
with 2 mM glutamine, 1% (v/v) heat inactivated human A–
B serum and 100 IU/mL of penicillin and streptomycin}.
PBMCs (100 lL) were cultured with test compounds
(50 lL) in tissue culture treated 96-well plates 30 min prior
to the addition of bacterial endotoxin lipopolysaccharide
(LPS) (E. coli type 0111:B4: 50 lL of 4 lg/mL) and then
incubated for a further ꢁ16 h. The production of TNFa in
response to LPS by the PBMCs was stopped by removal of
the cell medium from the PBMCs by aspiration. TNFa in
the cell assay medium was quantitated using a conven-
tional enzyme-linked immunosorbent assay and nonspe-
cific compound effects on PBMC viability measured by
incubation of the remaining treated cells in a WST-1 based
cytotoxicity assay.
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