Hepes and l glutamine

Peripheral blood mononuclear cells were isolated from healthy donor buffy coats (Sanquin, Amsterdam, The Netherlands) using Ficoll Paque Plus (GE Healthcare, Hoevelaken, The Netherlands) density centrifugation. Subsequently, monocytes were isolated by Percoll (GE Healthcare) density gradient as previously described (Repnik et al. 2003 (link)). Cells were cultured at 500,000 cells/ml in a 12-wells plate in RPMI-1640 (ThermoFisher Scientific) and adhered for 1 h and subsequently washed with warm phosphate-buffered saline (PBS). We then added IMDM medium containing HEPES and L-glutamine (Lonza) with 10% (v/v) FCS, 100 U/ml pen/strep and 20 ng/ml recombinant human M-CSF (PeproTech, London, UK). Cells were cultured for 8 days to differentiate to macrophages and subsequently treated with 100 ng/ml LPS for 18 h. As a pre-treatment we used salbutamol, norepinephrine and propranolol in dosages ranging from 0.1 μM to 10 μM. After 18 h, we collected supernatant for protein determination and macrophages were collected for RNA isolation.

FreeStyle Expi293F cells were cultured in FreeStyle 293 expression medium according to the manufacturer’s instructions (Invitrogen). Additional cell lines were obtained from the American Type Culture Collection (ATCC). Raji (human CD20-positive Burkitt’s lymphoma) cells were cultured in RPMI 1640 medium (Lonza), supplemented with 10% heat-inactivated Donor Bovine Serum with Iron (DBSI; Life Technologies), 4 mM L-glutamine, 25 mM Hepes and 1 mM Sodium Pyruvate (Lonza). Wien-133 (human CD52-positive Burkitt’s lymphoma) cells were cultured in Iscove’s Modified Dulbeco’s Medium (IMDM) with Hepes and L-glutamine (Lonza), supplemented with 10% heat-inactivated DBSI. All cell lines were maintained at 37°C in a 5% CO₂ humidified incubator.

Written informed consent was given by all women participating in this study. The study was approved by the ethics committee of the Faculty of Health Science (University of Abomey-Calavi) in Benin. The study was conducted at the Suru Léré maternity clinic, Cotonou, Benin. All women were tested for P. falciparum infection using a rapid diagnostic test (Parascreen, Zephyr Biomedicals Goa, India), and those with a positive result were included. P. falciparum IE were obtained from 123 pregnant women attending antenatal visit and 9 women admitted for delivery. Venous blood was collected in vacutainers with citrate phosphate dextrose adenine anticoagulant. Thick and thin blood films were prepared from blood samples to confirm P. falciparum infection. Hemoglobin values of women and the birth weight of their offspring were collected for all women included at delivery. Detailed characteristics of the study site have been previously described [33] (link).
Ring stage IE were allowed to mature in vitro to trophozoite-stage, as described [34] (link). Briefly, isolates were grown in RPMI 1640 supplemented with Hepes and L-glutamine (Lonza Biowhittaker), 0.3 g/L l-glutamine, 0.05 g/L gentamicin, 5 g/L albumax. Cultures were grown for no more than 48 h before testing. Ring stage parasites were also conserved in 10 volumes of TRIzol reagent (Invitrogen) and stored at −80°C until RNA extraction.