Inhibition of L. donovani axenic amastigotes Measured in Cell-Based System Using Plate Reader - 2138-04_Inhibitor_Dose_DryPowder_Activity
Expected Outcome: Compounds significantly suppressing Alamar blue, and L. donovani proliferation, are considered active. ..more
Keywords: Leishmania, L. donovani, MHOM-ET-67/L82
Leishmania donovani infected macrophagen assay
Expected Outcome: Compounds significantly suppressing Alamar blue, and L. donovani proliferation, are considered active.
Amastigotes of L. donovani strain MHOM/ET/67/L82 were grown in axenic culture at 37 degrees C in SM medium (Cunnigham et al. 1977) at pH 5.4 supplemented with 10% heat-inactivated fetal bovine serum under an atmosphere of 5% CO2 in air. One hundred microlitres of culture medium with 105 amastigotes from axenic culture with or without a serial drug dilution were seeded in 96-well microtitre plates. Serial drug dilutions of eleven 3-fold dilution steps covering a range from 100 to 0.002 ig/ml were prepared. After 70 h of incubation the plates were inspected under an inverted microscope to assure growth of the controls and sterile conditions. 10 il of Alamar Blue (12.5 mg resazurin dissolved in 100 ml distilled water) (Mikus and Steverdig. 2000) were then added to each well and the plates incubated for another 2 h. Then the plates were read with a Spectramax Gemini XS microplate fluorometer (Molecular Devices Cooperation, Sunnyvale, CA, USA) using an excitation wave length of 536 nm and an emission wave length of 588 nm. Data were analyzed using the software Softmax Pro (Molecular Devices Cooperation, Sunnyvale, CA, USA). Decrease of fluorescence (= inhibition) was expressed as percentage of the fluorescence of control cultures and plotted against the drug concentrations. From the sigmoidal inhibition curves the IC50 values were calculated by linear regression (Huber 1993). Miltefosine is used as control.
Standard parasite strains:
L. donovani MHOM-ET-67/L82, axenic amastigotes
Standart Host Cells:
Freshly harvested macrophages from NMRI, BalbC or CD1 mice
Miltefosin: average IC50 = 0.66
Medium: Mouse macrophages, dilution of parasites and drugs:
RPMI 1640 plus 10% heat inactivated FCS
Cultivation of axenic Leishmania
SM, pH 5.4 plus 10% heat inactivated FCS
Antibiotics: 1% MaserMix
Slides: Lab-tekTM 16-chamber Slides
Incubation: 37 degrees C, 5% CO2, 72 hours
Compounds are dissolved in DMSO at 10mg/ml (SOP Nr. D1). If insoluble other solvents are used according to the recommendations of the supplier. The DMSO stocks are kept at -20 degrees C. For the assays fresh dilutions in medium are prepared each time. (Since DMSO is toxic, care has to be taken not to exceed a final concentration of 1% DMSO in the assay).
Highest drug starting concentration: 10mg/ml
Day 1, Monday or Tuesday - Mouse inoculation
. One or two mice are inoculated i.p. with 2ml each of a 2% starch solution in distilled water (0.2g/10ml).
Day 2, Tuesday or Wednesday - Cell- harvesting
. Per Mouse prepare 10ml RPMI without FCS+1% MaserMix (100ml in 10ml) and put it on ice till usage.
Isolation of mouse macrophages: Euthanise mice using CO2 .Sterilise belly with 70% ethanol. Incise skin with a small cut and pull skin away from entire belly area. Wash again with ethanol. Lift skin over the sternum with forceps and inject 10ml of the prepared RPMI into the belly cavity. Grab the mouse by all four feet and swing gently in order to suspend the macrophages into the media. Lay the mouse on its back. Wash again with ethanol. Insert a 26 gauge needle on a syringe through the skin of the abdominal cavity. Enter low on the side to allow the fluid to pool. Extract medium slowly. Transfer into a tube and put it on ice.
. Centrifuge macrophages for 10 min at 1500rpm. Re-suspend pellet in 5 ml RPMI Medium.
. Determine cell concentration by haemocytometer. Dilute 1:10 in Trypanblue.
. Adjust cell concentration using RPMI +10%FCS +1% MaserMix to 4x105Cells/ml (4x104/well)
. 100ml of the 4x105/ml macrophage suspension are dispensed into each well of the 16-chamber slides (1.6ml needed per slide)
. Incubate for 24 hrs at 37 degrees C in 5% CO2
Day 3, Wednesday or Thursday - infection
. The macrophages are infected at a ratio of 1:3 (macrophages to amastigotes) with an axenic amastigote culture.
. Adjust concentration by diluting in RPMI +10%FCS to 1.2x106 /ml.
. Ad 100ml of the L. donovani suspension to each well.(1.6ml needed per slide)
. Mix contents of the wells carefully through pipetting.
. Incubate for 24hrs at 37 degrees C in 5% CO2
Preparation of amastigotes and cell count (CASY):
1. Draw medium with clumped amastigotes through a 1.2 x 40mm 18G (pink) needle into a syringe. Replace the needle with a 0.5 x 16mm 25G (yellow) needle and gently expel the liquid into a fresh Bijoux bottle. Repeat this process two more times, using fresh needles, syringe and Bijoux bottle every time.
2. Fill two CASY tubes with 10 ml isotone solution. Into the first tube (A), add 10 microl culture. From the second tube (B), remove 1 ml isotone solution, and replace with 1 ml solution from tube A. Measure tube B in CASY program 2.
3. Record counts/ml, but increase the power of 10 by 1 (CASY assumes that the dilution is 1:1000 but actually it's 1:10,000).
Day 4, Thursday or Friday - Drug treatment
. The degree of infection is checked by fixing (100% methanol, 5 min) and staining (10% Giemsa stain, 10 min) one slide for microscopic evaluation. Or, fix 1 well by adding 100ml methanol for 2-5 min, remove the methanol and add 20ml of Giemsa.
. For the other slides, the medium is removed and replaced by fresh RPMI 1640+10%FCS medium
. Mix media, remove again and add 200ml of fresh media.
. In a 48 Well Plate insert 300ml RPMI 1640+10%FCS medium per well and add 9ml of a 1mg/ml drug-stock-solution.(=3xhighest drug concentration)
. Add 100ml of pre-diluted drug from the 48 well-plate to the first set of wells in test slides.
. Mix and transfer 100ml into the following set of wells. Repeat this step for all wells, continuing onto the second test slide (1:3 serial drug dilution)
. Discard the last 100ml so that all wells contain 200ml.
. For the control slide, the first 8 wells are left without drugs. In the remaining 8 wells, standard drug (Miltefosin) is diluted longwise along the control slide (start conc. 1 mg/ml)
. Incubate for 96 hours at 37 degrees C in 5% CO2
Day 8, Monday or Tuesday - Staining
. The medium and the chambers are removed, the slides are fixed in 100% methanol for 5 min and stained with Giemsa (10%) for 10 min.
. The slides are then examined microscopically: the ratio of infected to uninfected macrophages is determined and the IC50 calculated by linear regression analysis.
Compounds that had IC50 less than 3 times the control compound, Miltefosine were labeled active.
* Activity Concentration.
Data Table (Concise)