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1st August 2013 @ 05:06

Results:

OSM-S-106 had a liver stage IC50 of 2.348 μM and OSM-S-111 had an IC50 of 345.4 nM. OSM-S-106 had no effect on the HepG2 host cells at the 10uM maximal concentration, while OSM-S-111 displayed some cytotoxicity with an IC50 of 4.386 μM.

Data:

OSM-S-106 and 111.xlsx

IC50 graphs.pdf

Materials & Methods

Parasites. P. berghei Luciferase sporozoites are obtained by dissection of infected A. stephensi mosquito salivary glands supplied by the New York University Insectary. Dissected salivary glands were homogenized in a glass tissue grinder and filtered twice through Nylon cell strainers (40 μm pore size, BD Falcon), spun down at 10,000xg, resuspended in media and counted using a hemocytometer. The sporozoites are diluted to a final concentration of 200 sporozoites per μl and kept on ice until needed.

Cell lines. HepG2-A16-CD81EGFP cells stably transformed to express a GFP-CD81 fusion protein (Silvie, O. 2006), are cultured at 37°C in 5% CO2 in DMEM (Invitrogen, Carlsbad, USA) supplemented with 10% FCS, 0.29 mg/ml glutamine, 100 units penicillin and 100 μg/ml streptomycin.

Sporozoite invasion assay. 3x103 HepG2-A16-CD81EGFP cells in 5μl of medium (2x105 cells/ml, 5%FBS, 5xPen/Strep/Glu) are seeded in 1536-well plates 20-26 hours prior to the actual infection. 18 hours prior to infection, 50nl of compound in DMSO (0.5% final DMSO concentration per well) are transferred with a PinTool (GNF Systems) into the assay plates (10 μM final concentration). Atovaquone and 0.5% DMSO are used as positive and negative controls, respectively. Penicillin and streptomycin are added to the sporozoite preparation for a final 5x-fold increased concentration in the well. The HepG2-A16-CD81EGFP cells were then infected with 103 sporozoites per well (5 μl) with a single tip Bottle Valve liquid handler (GNF), and the plates spun down at 37°C for 3 minutes in an Eppendorf 5810 R centrifuge with a centrifugal force of 330x on lowest acceleration and brake setting. After incubation at 37°C for 48 hours the EEF growth was quantified by bioluminescence.

Bioluminescence quantification of exo-erythrocytic forms (EEFs). Media is removed by spinning the inverted plates at 150xg for 30 seconds. 2 μl BrightGlo (Promega) are dispensed with the MicroFlo (BioTek) liquid handler. Immediately after addition of the luminescence reagent, plates are vortexed at the median intensity setting for 10 seconds and read by the Envision Multilabel Reader (PerkinElmer). IC50 values are obtained using measured bioluminescence intensity and a non-linear variable slope four parameter regression curve fitting model in Prism 6 (GraphPad Software Inc). 


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Attached Files
OSM-S-106 and 111.xlsx
IC50 graphs.pdf