Inhibition of Trypanosoma brucei rhodesiense STIB900 Measured in Cell-Based System Using Plate Reader - 2138-05_Inhibitor_Dose_CherryPick_Activity
Expected Outcome: Compounds significantly suppressing Alamar blue, and thus T. brucei proliferation, are considered active. ..more
BioActive Compounds: 2
Keywords:T. brucei, Trypanosoma, HAT, african trypanosomiasis
Assay Overview: Activity against Trypanosoma brucei rhodesiense STIB900.
Expected Outcome: Compounds significantly suppressing Alamar blue, and thus T. brucei proliferation, are considered active.
This stock was isolated in 1982 from a human patient in Tanzania and after several mouse passages cloned and adapted to axenic culture conditions (Baltz et al 1985) Minimum Essential Medium (50 microl) supplemented with 25 mM HEPES, 1g/l additional glucose, 1% MEM non-essential amino acids (100x), 0.2 mM 2-mercaptoethanol, 1mM Na-pyruvate and 15% heat inactivated horse serum was added to each well of a 96-well microtiter plate. Serial drug dilutions of eleven 3-fold dilution steps covering a range from 100 to 0.002 ig/ml were prepared. Then 4x103 bloodstream forms of T. b. rhodesiense STIB 900 in 50 microl was added to each well and the plate incubated at 37 degrees C under a 5 % CO2 atmosphere for 70 h. 10 microl Alamar Blue (resazurin, 12.5 mg in 100 ml double-distilled water) was then added to each well and incubation continued for a further 2-4 h (Raz et al 1997). 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. The IC50 values were calculated by linear regression (Huber 1993) from the sigmoidal dose inhibition curves using SoftmaxPro software (Molecular Devices Cooperation, Sunnyvale, CA, USA). Melarsoprol is used as control.
Baltz, T., D. Baltz, C. Giroud, and J. Crockett. 1985. Cultivation in a semi-defined medium of animal infective forms of Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesiense and T. gambiense. EMBO Journal 4:1273-1277.
Raz, B., M. Iten, Y. Grether-Buhler, R. Kaminsky, and R. Brun. 1997. The Alamar Blue assay to determine drug sensitivity of African trypanosomes (T.b. rhodesiense and T.b. gambiense) in vitro. Acta Trop 68:139-47.
Huber, W. Koella, J.C. 1993. A comparison of the three methods of estimating EC50 in studies of drug resistance of malaria parasites. Acta Trop. 55, 257-261.
In vitro sensitivity assays: African Trypanosomes (LILIT, Alamar Blue)
Standard Parasite Strains:
Trypanosoma brucei rhodesiense; STIB 900
MelarsoprolTM (Main standard for STIB 900)
Pentamidine (PentacarinatTM, Rhone-Poulenc, France)
Suramine (GeramineTM, Bayer, Germany)
Medium: T.b. rhodesiense, T.b. brucei
1mL Balz-components (Balz et al., 1985)
2-Mercaptoethanol: 1mL of a diluted stock (14mL 2-mercaptoethanol + 10mL dH2O)
15mL heat inactivated horse serum (15% final concentration)
MEM supplemented with Balz-components and 2-Mercaptoethanol
+ 5% heat inactivated foetal calf serum + 15% human serum
Dulbeccos medium modified by Iscoves supplemented + 20% heat
inactivated goat serum
Plates: CostarTM 96-well microtitre plates
Incubation: 37 degrees C, 5% CO2
Definition of test score:
inactive (no repeat): IC50 > 3mg/ml
moderate activity (repeat): 0.2mg/ml < IC50 < 3mg/ml
high activity (repeat): IC50 < 0.2mg/ml (for active series<0.1)
Melarosprol (in STIB 900): average IC50 = 0.004
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).
1. Into the wells of row H, add 75microl of medium that contains two times the highest drug concentration desired. Per plate 4 drugs can be tested (drug 1 column 1-3, drug 2 column 4-6, etc.)
2. Add 50 microl of medium at room temperature to rows A to G of a 96-well plate (row H has the drug).
3. Serial drug dilutions are prepared by using a 12-well multi-pipette. First, remove 25 microl from wells of row H and put it into row G and mix well. Next, 25 microl are taken out of row G and put into row F and so on until row B. The last 25 microl of row B are discarded. A serial dilution factor of 1:3 is thus obtained. Row A wells serve as controls without drugs.
4. 50 microl of medium without trypanosomes are added to columns 3, 6, 9 and 12; these columns serve as background controls (that may be caused by the drug).
5. Dilute the trypanosomes to 3 x 104 tryps/ml. The trypanosome density is adjusted with a Cell Analysis System (CASY, Scharfe System) or by a count on the haemocytometer. (The trypanosome density used should be adjusted depending on the current growth characteristics of the corresponding cultures)Per plate, allow for the use of 3.5ml of the trypanosome stock.
6. Into the remaining wells, add 50 microl of trypanosome suspension.
7. The plates are then incubated for 69h (= 72h - time incubated with Resazurin)* at 37 degrees C / 5% CO2.
1. The plates are inspected under an inverted microscope to ensure that growth is normal. Additional information may be recorded, such as drug insolubility or contamination, etc.
2. Add 10 microl of the fluorescent dye Resazurin to each well and incubate for an additional 3 hours (until a subtle color change is observed, but maximum 5 hours)*.
3. To determine an IC50 value, the plate is read at excitation wavelength 530 nm and emission wavelength 590 nm (pre-set LILIT template file). Make sure that the values in each well are approximately 10 times the background values.
4. Data are transferred into a graphic program (Excel) and are evaluated to determine the IC50 or analyzed using the fluorescent plate reader software (SoftMax).
* These are recommended times for STIB 900. When working with other strains, adjust incubation times so that an appropriate color change is achieved, while maintaining the assay duration of 72 hours
Raz B, Iten M, Grether-Buhler Y, Kaminsky R, and Brun R. (1997). The Alamar Blue assay to determine drug sensitivity of African trypanosomes in vitro. Acta Trop. 68:139-147
Categorized Comment - additional comments and annotations
* Activity Concentration.
Data Table (Concise)