Lipopolisaccaride lps

PBMC were obtained from buffy coats of blood of healthy donors using a Ficoll-Paque density gradient centrifugation and monocytes were purified using negative selection antibody cocktails (StemCell Technologies) as described before [22 (link)]. Monocytes were cultured in complete culture medium (RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco) or human serum (HS; Sigma) and penicillin/streptomycin (Gibco) and differentiated to monocyte derived macrophages (MDM) for 4 days in the presence of monocyte-colony stimulating factor (M-CSF, Peprotech) or granulocyte-macrophage colony-stimulating factor (GM-CSF, Peprotech) both at 100 ng/ml. The protocol was approved by the scientific committee of Fundació IrsiCaixa. Buffy coats were purchased from the Catalan Banc de Sang i Teixits (http://www.bancsang.net/en/index.html). The buffy coats received were totally anonymous and untraceable and the only information given was whether or not they have been tested for disease. When appropriate, differentiated macrophages were incubated with 100 ng/ml of lipopolisaccaride (LPS, Sigma-Aldrich) overnight at 37°C.
TZM cells were received from the National Institutes of Health, AIDS Research and Reference Reagent Program. HEK293T cells were purchased from Dharmacon (Madrid, Spain).

3-Azido-3-deoxythymidine (zidovudine, AZT) was purchased from Sigma-Aldrich (Madrid, Spain). Raltegravir (RAL) was obtained from the NIH AIDS Research and Reference Reagent Program. MRT67307, a pharmacological inhibitor of IKKϵ and TBK1, was purchased from Selleckchem. When appropriate, differentiated macrophages were incubated with 100 ng/ml of lipopolisaccaride (LPS, Sigma-Aldrich) or 10 µg/ml of Poly I:C (Sigma-Aldrich), during4 hours at 37 °C.