After written informed consent was provided, human donor blood was
obtained at the National Institutes of Health (NIH) Clinical Center under
Institutional Review Board (IRB)–approved protocols. PBMCs were purified
from blood by Ficoll (Fisher Scientific) density gradient centrifugation, washed
with RPMI 1640 plus penicillin, streptomycin, and L-glutamine (Gibco) along with
10% FBS (Sigma-Aldrich). Cells were resuspended to a concentration of 1
× 106 cells/ml and aliquoted (1 ml/well) into 48-well plates.
LPS (0.5 μg/ml Sigma-Aldrich) and brefeldin A (5 μg/ml,
Sigma-Aldrich) were added before incubation at 37 °C overnight. After
incubation, cells were washed with FACS buffer and stained with LIVE/DEAD Blue
(Life Technologies). Cells were then fixed in 1.6% paraformaldehyde for
10 min at room temperature, spun down, resuspended in 100% cold
methanol, and incubated overnight at −20 °C. Cells were then
washed and stained with fluorochrome-conjugated antibodies: CD14 (MφP9,
BD Biosciences), CD11c (B-ly6, BD Biosciences), Ikaros (R32-1149, BD
Biosciences), IL-1β (AS10, BD Biosciences), TNF-α (MAb11, BD
Biosciences), and IL-6 (MQ2-13A5, eBioscience). The data were gated on
CD11c+CD14+ monocytes.
obtained at the National Institutes of Health (NIH) Clinical Center under
Institutional Review Board (IRB)–approved protocols. PBMCs were purified
from blood by Ficoll (Fisher Scientific) density gradient centrifugation, washed
with RPMI 1640 plus penicillin, streptomycin, and L-glutamine (Gibco) along with
10% FBS (Sigma-Aldrich). Cells were resuspended to a concentration of 1
× 106 cells/ml and aliquoted (1 ml/well) into 48-well plates.
LPS (0.5 μg/ml Sigma-Aldrich) and brefeldin A (5 μg/ml,
Sigma-Aldrich) were added before incubation at 37 °C overnight. After
incubation, cells were washed with FACS buffer and stained with LIVE/DEAD Blue
(Life Technologies). Cells were then fixed in 1.6% paraformaldehyde for
10 min at room temperature, spun down, resuspended in 100% cold
methanol, and incubated overnight at −20 °C. Cells were then
washed and stained with fluorochrome-conjugated antibodies: CD14 (MφP9,
BD Biosciences), CD11c (B-ly6, BD Biosciences), Ikaros (R32-1149, BD
Biosciences), IL-1β (AS10, BD Biosciences), TNF-α (MAb11, BD
Biosciences), and IL-6 (MQ2-13A5, eBioscience). The data were gated on
CD11c+CD14+ monocytes.