Bookmark and Share
BioAssay: AID 651881

Inhibition 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_Activity

Assay Overview: Rat skeletal myoblasts (L-6 cells) were infected with T. cruzi Tulahuen strain C2C4 containing the a-galactosidase (Lac Z) gene (Buckner et al. 1996). After 48 hr, extracellular T. cruzi were removed and cells were treated with or without compounds. After 96 h of incubation the plates were inspected under an inverted microscope to assure growth of the controls and sterility. a-galactosidase, and thus T. cruzi proliferation, was measured by additon of TCPRG/Nonidet. Wells were read photometrically at 540 nm within 2-6 hrs of read reagent additon. ..more
_
   
 Tested Compounds
 Tested Compounds
All(20)
 
 
Active(5)
 
 
Inactive(15)
 
 
 Tested Substances
 Tested Substances
All(20)
 
 
Active(5)
 
 
Inactive(15)
 
 
 Related BioAssays
 Related BioAssays
AID: 651881
Data Source: Broad Institute (2138-03_Inhibitor_Dose_CherryPick_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-12-06
Hold-until Date: 2012-12-08
Modify Date: 2012-12-08

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 5
Related Experiments
Show more
AIDNameTypeProbeComment
624280Broad Institute Inhibition of T.cruzi replication in culture Inhibitor Probe ProjectSummary1 depositor-specified cross reference: Summary assay
624255Inhibition of T.cruzi proliferation in culture Measured in Cell-Based System Using Plate Reader - 2138-01_Inhibitor_SinglePoint_HTS_ActivityScreening same project related to Summary assay
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
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
651890Inhibition 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_DryPowder_ActivityConfirmatory same project related to Summary assay
651891Inhibition of L. donovani axenic amastigotes Measured in Cell-Based System Using Plate Reader - 2138-04_Inhibitor_Dose_CherryPick_ActivityConfirmatory same project related to Summary assay
651892Inhibition of L. donovani axenic amastigotes Measured in Cell-Based System Using Plate Reader - 2138-04_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
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
651894Inhibition of Trypanosoma brucei rhodesiense STIB900 Measured in Cell-Based System Using Plate Reader - 2138-05_Inhibitor_Dose_DryPowder_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
652279Cidal 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_ActivityOther same project related to Summary assay
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
686919Inhibition of Trypanosoma cruzi Cruzain Measured in Biochemical System Using Plate Reader - 2138-09_Inhibitor_Dose_DryPowder_ActivityOther same project related to Summary assay
Description:
Keywords: T. cruzi, Tulahuen, amastigotes, Trypanosoma cruzi


Assay Overview: Rat skeletal myoblasts (L-6 cells) were infected with T. cruzi Tulahuen strain C2C4 containing the a-galactosidase (Lac Z) gene (Buckner et al. 1996). After 48 hr, extracellular T. cruzi were removed and cells were treated with or without compounds. After 96 h of incubation the plates were inspected under an inverted microscope to assure growth of the controls and sterility. a-galactosidase, and thus T. cruzi proliferation, was measured by additon of TCPRG/Nonidet. Wells were read photometrically at 540 nm within 2-6 hrs of read reagent additon.

Expected Outcome: Compounds significantly suppressing CPRG colorometric assays, and therefore a-galactosidase expression will be identified as hits in the screen. Compounds that inhibit color development of CPRG or are toxic to the host cell will also be identified as actives.
Protocol
Protocol:
Assay Description: Rat skeletal myoblasts (L-6 cells) were seeded in 96-well microtitre plates at 2000 cells/well in 100 iL RPMI 1640 medium with 10% FBS and 2 mM l-glutamine. After 24 h the medium was removed and replaced by 100 il per well containing 5000 trypomastigote forms of T. cruzi Tulahuen strain C2C4 containing the a-galactosidase (Lac Z) gene (Buckner et al. 1996). After 48 h the medium was removed from the wells and replaced by 100 il fresh medium with or without a serial drug dilution of eleven 3-fold dilution steps covering a range from 100 to 0.002 ig/ml. After 96 h of incubation the plates were inspected under an inverted microscope to assure growth of the controls and sterility. Then the substrate CPRG/Nonidet (50 il) was added to all wells. A color reaction developed within 2-6 h and could be read photometrically at 540 nm. Data were analyzed with the graphic programme Softmax Pro (Molecular Devices), which calculated IC50 values by linear regression (Huber 1993) from the sigmoidal dose inhibition curves. Benznidazole is used as control.

Standard assay parasite strains:
T. cruzi Tulahuen C2C4, containing the Lac Z gene.
Plasmid construct by Dr. S. Reed
Received from Dr. Buckner, UW, Seattle as epimastigotes in LIT, transformed by E.G.

Standard Cell line:
L-6 cells (mouse muscle fibroblasts)

Standard Drug:
Benznidazole (RadanilTM, Hoffman La Roche): start conc. 30microg/ml (IC50 = 0.35 microg/ml)

Standard Conditions:
Medium: RPMI 1640 + 10% FCS + 1.7microM L-Glutamine (850microl 200mM for 100ml)

Plates: CostarTM 96-well microtiter plates

Incubation: 370C, 5% CO2

Substrate: 2.5X CPRG/Nonidet Solution:
5X stock = 500 ml Nonidet P40+ 30.38mg CPRG in 100 ml 1X PBS
Dilute the 5X stock 1:1 with 1X PBS.
Light Sensitive!

Definition of test score:
inactive (no repeat): IC50 > 30mg/mL
moderate activity (repeat): 2mg/mL < IC50 < 30mg/mL
high activity (repeat): IC50 < 2mg/mL (active series < 1mg/mL)
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).

Procedure:

Day 1:
Seed all 96 wells with 100 microl medium containing 2x103 L6 cells per well, using a multi-well repeater pipette.

9.6 ml per plate
2x104 L-6 cells/ml....100 microl per well

Day 2:
Add 5x103 tryps into all columns 1 and 2, 4 and 5, 7 and 8, and 10 and 11 using the multi-well repeater pipette. In columns 3, 6, 9 and 12, add 50 microl of medium.

3.2 ml of trypanosome suspension per plate
1x105 tryps/ml.......50 microl per well

Day 4:
Remove medium from wells in row A to G with the aspirator and replace with 100microl medium using the multi-well repeater pipette. (Take care to not cross infect columns 3, 6, 9 and 12-remove medium only before medium + tryps!)

Remove medium from row H (do not cross-infect!) and replace with 150 microl of the highest drug concentration. Per plate 4 drugs can be tested (drug 1 columns 1-3, drug 2 columns 4-6, etc.). Note: Do the first half of the plates (remove medium and add drug) and then the second half, so the cells/tryps don't dry out.

Serial drug dilutions are prepared by using a 12-channel multi-pipette. First, remove 50 microl from wells of row H and put into row G and mix well. Next, 50 microl are taken out of row G and put into row F and so on until row B. The last 50 microl of row B are discarded. A serial dilution factor of 1:3 is thus obtained. Wells in row A serve as control wells without drugs.

Day 8:
Evaluate the plates visually to determine the MIC (Minimal Inhibitory Concentration): lowest drug concentration at which no trypanosomes with a normal morphology and motility as compared to the control wells can be seen. Additional information may be recorded, such as drug insolubility or contamination, etc. Also, preliminary cytotoxicity (MIC) is to be noted in columns 3, 6, 9 and 12.

50 microl of 2.5X CPRG/ Nonidet are added to all wells. A color reaction will become visible in 2-6 hours and can be read in an Absorbance Reader at 540nm.

Data are transferred into a graphic program (e.g. Excel) and IC50 values are calculated.
Comment
EXPECTED OUTCOME: Compounds with IC50 less than 25 fold the control compound, Chloroquine, were labeled active.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Format: Cell-based
Assay Type: Functional
Assay Cell Type: L6
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
PageFrom: