Human Derived Macrophages obtained from four healthy donors and individually infected with Leishmania major metacyclic parasites
Biomaterial provider
Fatma Z. Guerfali*†, Dhafer Laouini*†, Lamia Guizani-Tabbane*†, Florence Ottones§†, Khadija Ben-Aissa*†, Laurent Manchon¶, David Piquemal¶, Jacques Marti§†, and Koussay Dellagi. *LIVGM. Institut Pasteur de Tunis, Tunisia.†Laboratoire International Associé, CNRS, France, §Groupe d’Etude des Transcriptomes (GET). Institut de Génétique Humaine, UPR CNRS 1142, Montpellier, France.¶Skuld-Tech, 88, Montpellier, 34080, France.
Treatment protocol
Human PBMC were isolated from leukopacks peripheral blood mononuclear cells of four healthy volunteers using Ficoll-Paque density gradient centrifugation. Cells were washed and resuspended at 106 cells/ml in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 10% autologous heat inactivated serum. Monocytes were purified by fibronectin-mediated adhesion using gelatin and autologous heat inactivated serum substratum.
Growth protocol
In order to obtain macrophages, monocytes were cultured at 37°C, 5% CO2 in endotoxin-free RPMI 1640 medium supplemented with 10% heat-inactivated normal human AB serum and 10% heat-inactivated fetal calf serum (HyClone Laboratories, Logan, UT), 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine for 7 days at 2 x 106 cells/ml, in 6-well tissue-culture plates. Obtained MFs were exposed to metacyclic parasites of L. major (MHOM/TN/95/GLC94 strain (ratio parasites:cells 5:1) for 24h and then harvested for RNA preparation.
Extracted molecule
polyA RNA
Extraction protocol
Cells or parasites were collected at the indicated time points by centrifugation, homogenized by Trizol reagent (Gibco BRL) and frozen at -70°C until RNA extraction. The RNA from each of the 4 donors was extracted independently then pooled, and used for library construction. RNA was purified of contaminating genomic DNA by using standard protocol. Briefly, contaminating DNA was removed from total macrophage or parasite RNA by using DNase I (Invitrogen, Carlsbad, Calif). The RNA samples were then ethanol-precipitated, washed once in 70% ethanol, and re-dissolved in water. RNA was quantified using spectrophotometer. Examination of purified total RNA by gel electrophoresis revealed prominent 5S, 18S and 28S ribosomal bands for human samples and 18S and 24Sa and 24Sb ribosomal bands for parasitic samples indicating that the RNA was not degraded.
Description
Human Monocyte Derived Macrophage infected with Leshmania major parasites
