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BioAssay: AID 651894

Inhibition of Trypanosoma brucei rhodesiense STIB900 Measured in Cell-Based System Using Plate Reader - 2138-05_Inhibitor_Dose_DryPowder_Activity

Expected Outcome: Compounds significantly suppressing Alamar blue, and thus T. brucei proliferation, are considered active. ..more
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 Tested Compounds
 Tested Compounds
All(8)
 
 
Active(2)
 
 
Inactive(6)
 
 
 Tested Substances
 Tested Substances
All(8)
 
 
Active(2)
 
 
Inactive(6)
 
 
 Related BioAssays
 Related BioAssays
AID: 651894
Data Source: Broad Institute (2138-05_Inhibitor_Dose_DryPowder_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2012-12-07
Hold-until Date: 2013-07-15
Modify Date: 2013-07-16

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 2
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624280Broad Institute Inhibition of T.cruzi replication in culture Inhibitor Probe ProjectSummary1 depositor-specified cross reference: Summary Assay
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651739Inhibition of T.cruzi proliferation in culture Measured in Cell-Based System Using Plate Reader - 2138-01_Inhibitor_SinglePoint_CherryPick_ActivityOther same project related to Summary assay
651740Inhibition of T.cruzi proliferation in culture Measured in Cell-Based System Using Plate Reader - 2138-01_Inhibitor_SinglePoint_CherryPick_Activity_Set2Other same project related to Summary assay
651742NIH/3T3 (mouse embryonic fibroblast) toxicity Measured in Cell-Based System Using Plate Reader - 2138-02_Inhibitor_SinglePoint_CherryPick_ActivityOther same project related to Summary assay
651744NIH/3T3 (mouse embryonic fibroblast) toxicity Measured in Cell-Based System Using Plate Reader - 2138-02_Inhibitor_SinglePoint_CherryPick_Activity_Set2Other same project related to Summary assay
651817NIH/3T3 (mouse embryonic fibroblast ) toxicity Measured in Cell-Based System Using Plate Reader - 2017-02_Inhibitor_Dose_CherryPick_Activity_Set2Confirmatory same project related to Summary assay
651818Intracellular Trypanosomes Measured in Cell-Based/Microorganism System Using Plate Reader - 2017-01_Inhibitor_Dose_CherryPick_Activity_Set3Confirmatory same project related to Summary assay
651844NIH/3T3 (mouse embryonic fibroblast) toxicity Measured in Cell-Based System Using Plate Reader - 2138-02_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651845Inhibition of T.cruzi proliferation in culture Measured in Cell-Based System Using Plate Reader - 2138-01_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651869Cidal vs. static assay: inhibition of T. cruzi (CAI/72) after prolonged treatment and treatment withdrawal Measured in Cell-Based System Using Imaging - 2138-08_Inhibitor_SinglePoint_DryPowder_Activity_Set2Other same project related to Summary assay
651877Cidal vs. static assay: inhibition of T. cruzi (CAI/72) after prolonged treatment and treatment withdrawal Measured in Cell-Based System Using Imaging - 2138-08_Inhibitor_SinglePoint_DryPowder_ActivityOther same project related to Summary assay
651881Inhibition of T. cruzi Tulahuen strain amastigotes in L-6 muscle cells Measured in Cell-Based and Microorganism Combination System Using Plate Reader - 2138-03_Inhibitor_Dose_CherryPick_ActivityConfirmatory same project related to Summary assay
651885Inhibition of intracellular CAI/72 strain of Trypanosoma cruzi in bovine embryo skeletal muscle cells: high content imaging Measured in Cell-Based System Using Imaging - 2138-07_Inhibitor_SinglePoint_DryPowder_ActivityConfirmatory same project related to Summary assay
651888Inhibition of Plasmodium falcipirum NF54 Measured in Cell-Based System Using Plate Reader - 2138-11_Inhibitor_Dose_CherryPick_ActivityConfirmatory same project related to Summary assay
651889Inhibition of Plasmodium falcipirum NF54 Measured in Cell-Based System Using Plate Reader - 2138-11_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
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651893Inhibition of Trypanosoma brucei rhodesiense STIB900 Measured in Cell-Based System Using Plate Reader - 2138-05_Inhibitor_Dose_CherryPick_ActivityConfirmatory same project related to Summary assay
651895L-6 rat myocyte cell toxicity Measured in Cell-Based System Using Plate Reader - 2138-06_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651896L-6 rat myocyte cell toxicity Measured in Cell-Based System Using Plate Reader - 2138-06_Inhibitor_Dose_CherryPick_ActivityConfirmatory same project related to Summary assay
651897Inhibition of Trypanosoma cruzi Cruzain Measured in Biochemical System Using Plate Reader - 2138-09_Inhibitor_SinglePoint_DryPowder_ActivityConfirmatory same project related to Summary assay
651903Intracellular Trypanosomes Measured in Cell-Based/Microorganism System Using Plate Reader - 2017-01_Inhibitor_SinglePoint_HTS_Activity_Set2Screening same project related to Summary assay
652275Inhibition of T.cruzi proliferation in culture Measured in Cell-Based System Using Plate Reader - 2138-01_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
652278Cidal vs. static assay: inhibition of T. cruzi (CAI/72) in J774 macrophages after prolonged compound treatment and treatment withdrawal Measured in Cell-Based System Using Imaging - 2138-13_Inhibitor_SinglePoint_DryPowder_Activity_Set2Other same project related to Summary assay
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652280NIH/3T3 (mouse embryonic fibroblast) toxicity Measured in Cell-Based System Using Plate Reader - 2138-02_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
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Description:
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.
Protocol
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)
Aug. 2007


Standard Parasite Strains:
Trypanosoma brucei rhodesiense; STIB 900
Standard Drug:
MelarsoprolTM (Main standard for STIB 900)
Pentamidine (PentacarinatTM, Rhone-Poulenc, France)
Suramine (GeramineTM, Bayer, Germany)


Standard Conditions:
Medium: T.b. rhodesiense, T.b. brucei
Per 100mL:
83mL MEM
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)

T.b. gambiense
MEM supplemented with Balz-components and 2-Mercaptoethanol
+ 5% heat inactivated foetal calf serum + 15% human serum

T. congolense
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


Drug preparation:
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.


Evaluation
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

References:
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
Comment
Compounds that had IC50 less than 1 uM were labeled active.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_uM *FloatμM

* Activity Concentration.
Additional Information
Grant Number: 1 R03 MH085673-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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