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MB Sample ID: SA219001
| Local Sample ID: | IL04_U_0_CETOF |
| Subject ID: | SU002371 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens + Leishmania donovani |
| Taxonomy ID: | 9606 and 5661 |
| Genotype Strain: | Human: PBMCs from 7 human donors. L. donovani: MHOM/IN/82/Patra1 and MHOM/ET/1967/Hu3 |
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Collection:
| Collection ID: | CO002364 |
| Collection Summary: | Cloned lines of Leishmania donovani (MHOM/IN/82/Patra1 and MHOM/ET/1967/Hu3) were maintained with weekly subpassages at 27 ºC in complete RPMI 1640 medium, supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin plus 100 mg/mL streptomycin and 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer. Only parasites under ten passages were used in the experimental work. Peripheral blood mononuclear cells (PBMCs) were enriched from blood from healthy volunteers using a density gradient with Histopaque 1077. The mononuclear phase was recovered and washed with PBS. Next, cells were labeled with magnetic CD14 MicroBeads and CD14+ monocytes were positively separated using an MS column. Human CD14+ monocytes were differentiated in macrophages using recombinant macrophage colony stimulating factor 1 (M-CSF). Briefly, monocytes were plated at 1·106 cells/mL with 20 ng/mL of M-CSF. Growth factor was renewed at day 4 post-differentiation. The macrophages were used 7 days after differentiation. Macrophages were infected with L. donovani promastigotes at a 1:10 ratio. After 4 hours of incubation, non-phagocytosed parasites were removed, and cells were recovered. Macrophages were left in culture for 12, 36 and 72 h for metabolomic analysis. Uninfected macrophages were used as a control. To isolate uninfected and L. donovani-infected human monocyte-derived macrophages for further metabolomic analyses, cells were recovered, washed with phosphate buffer saline (PBS) and transferred into a previously weighted Eppendorf. After centrifugation the supernatant was discarded, and the pellet was immediately frozen in liquid nitrogen and stored at -80 ºC. |
| Sample Type: | Cultured cells |
