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BioAssay: AID 651739

Inhibition of T.cruzi proliferation in culture Measured in Cell-Based System Using Plate Reader - 2138-01_Inhibitor_SinglePoint_CherryPick_Activity

Assay Overview: The assay detects intracellular trypanosomes that are replicating inside host cells. NIH3T3 cells are trypsinized, counted, and plated in 384-well tissue culture plates. After plating the NIH3T3 cells, compounds are added to the wells. T. cruzi that are genetically modified to express b-galactosidase are then harvested and active Trypomastigotes form are counted and co-cultured with the NIH3T3 cells. After 90 hours of co-culture, all cells in the well are lysed and b-galactosidase production is detected using a luminescent reporter system (GalScreen). ..more
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 Tested Compounds
 Tested Compounds
All(5589)
 
 
Active(4045)
 
 
Inactive(1324)
 
 
Inconclusive(223)
 
 
 Tested Substances
 Tested Substances
All(5600)
 
 
Active(4051)
 
 
Inactive(1326)
 
 
Inconclusive(223)
 
 
 Related BioAssays
 Related BioAssays
AID: 651739
Data Source: Broad Institute (2138-01_Inhibitor_SinglePoint_CherryPick_Activity)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-10-31

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 4045
Related Experiments
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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
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
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: Trypanosoma cruzi, Chagas disease

Assay Overview: The assay detects intracellular trypanosomes that are replicating inside host cells. NIH3T3 cells are trypsinized, counted, and plated in 384-well tissue culture plates. After plating the NIH3T3 cells, compounds are added to the wells. T. cruzi that are genetically modified to express b-galactosidase are then harvested and active Trypomastigotes form are counted and co-cultured with the NIH3T3 cells. After 90 hours of co-culture, all cells in the well are lysed and b-galactosidase production is detected using a luminescent reporter system (GalScreen).

Expected Outcome: Compounds significantly suppressing luminescence, and therefore b-galactosidases expression will be identified as hits in the screen. Compounds that inhibit luminescence activity may kill T. cruzi, inhibit T. cruzi invasion or inhibit development of the parasite within the host cell. Compounds that are toxic to the host cell will be excluded in secondary assays.
Protocol
PROTOCOL: HTS ASSAY in 384-well plate TRYPANOSOMA CRUZI

A. Cells and parasite:
-LLC-MK2 cells (rhesus monkey kidney epithelial cell line): ATCC CCL7.1
-NIH/3T3 cells (mouse embryonic fibroblast cell line): ATCC CRL-1658
-Trypanosoma cruzi expressing beta-galactosidase (Tc-beta-gal: Tulahuen strain, clone C4 from: Buckner et al., 1996, Antimicrobial agents and chemotherapy, 40:11, pp 2592-2597.): ATCC PRA-330
C. Reagents, materials, solutions and culture media:
Reagents:
-DMEM with Phenol Red, high glucose, with L-glutamine and Sodium pyruvate (Cellgro, cat number: 10-013-CM)
-PSG or Penicillin-streptomycin-L-glutamine (Gibco-Invitrogen, cat number: 10378-016)
-FBS-Heat inactivated fetal bovine serum (Gibco-invitrogen, cat number: 16140-089)
-0.25% Trypsin-EDTA 1X (Gibco-Invitrogen, cat number: 25200-072)
-Sterile PBS (Phosphate Buffer Saline) 1X
- GalScreen, Buffer B (Applied Biosystems, cat number: T1031)
- Horse Serum (Invitrogen, 16050-114)
Materials:
-T225 culture flasks with vented caps (BD Falcon, cat number: 353028)
- Corning Hyperflasks (Corning, cat number: 10024)
Culture medium:
-For cell propagation:
90% DMEM+Phenol red, 10% FBS, 1% PSG. Mix and filter through 0.2 microns membrane. Keep at 4 degrees C. Warm up to 37 degrees C in water bath before use.
-For Tc culture and assays:
98% DMEM+Phenol red, 2% FBS, 10% Horse Serum, 1% PSG. Mix and filter through 0.2 microns membrane. Keep at 4 degrees C. Warm up to 37 degrees C in water bath before use.
98% DMEM+Phenol red, 2% FBS, 1% PSG. Mix and filter through 0.2 microns membrane. Keep at 4 degrees C. Warm up to 37 degrees C in water bath before use.

Solutions:
-GalScreen . Using GalScreen base kit (Applied Biosystems), mix Buffer B (Applied Biosystems, T2361) with 1:25 substrate (Applied Biosystems, T2359) .

D. Parasite and cell culture
Cell culture : NIH/3T3
Notes:
-NIH/3T3 cells are cultivated in DMEM supplemented with 10% FBS and 1% PSG in T225.
-Conditions can be adapted to other culture plate sizes.
-Cells are usually passaged
Protocol:
-Aspirate medium.
-Rinse cells with 10 ml PBS/plate.
-Aspirate PBS.
-Add 4 ml of pre-warmed trypsin-EDTA, swirl the dish to make sure the trypsin covers all the cells.
-Leave the dish a few minutes at room temperature (usually not more than 5).
-Check that the cells are detaching.
-Add 21 ml of medium, pipet up and down to detach all the cells.
-Take 75 ul of media and add to Cellometer cassette (Nexcelom Biosciences). Using the Cellometer Auto T4 (Nexcelom Biosciences), focus so cell bodies have dark edges and clear/white centers. Select NIH3T3 preset menu, dilution 1x, display image, and count. Using this number dilute in either 2% FBS/DMEM for assays or 10% FBS/DMEM for propagation
Cell culture : LLC-MK2
Notes:
-LLC-MK2 cells are cultivated in DMEM +Phenol red supplemented with 10% FBS and 1% PSG in T175 flasks in 35 ml total of medium.
-Conditions can be adapted to other culture plate sizes.
-Cells are usually passaged twice a week at 1:4 to 1:8 ratios.
Protocol:
-Aspirate medium.
-Rinse cells with 10 ml PBS/plate.
-Aspirate PBS.
-Add 5 ml of pre-warmed trypsin-EDTA, swirl the dish to make sure the trypsin covers all the cells.
-Place the dishes back in the incubator for 5 min.
-Check that the cells are detaching.
-Add 20 ml of medium, pipet up and down to detach all the cells. Dilute into 2% FBS/DMEM for assay or 10% FBS/DMEM for propagation.
Parasite culture: Trypanosoma cruzi beta-gal (Tc)
Notes:
-Tc-beta-gal are cultivated in DMEM +Phenol red supplemented with 2% FBS and 1% PSG in T175 flasks with vented caps in 35 ml total of medium.
Protocol:
-The day before infection (or at least 2-3 hours before), plate 2.5 Million LLC-MK2 cells/T175 in DMEM+2% FBS
-Either thaw Tc in from liquid nitrogen stock into 50 ml 2% FBS/DMEM or remove the media from a propagating LLC-MK2 co-culture, in other words, harvesting the medium containing the free Tc in 50 ml falcon tubes.
-Spin 10 min at 2200 rpm
-Aspirate the supernatant until 15 ml is left
-Place back the tubes in the incubator delicately (so as not to disturb the pellet)
-Incubate minimum 4 hours at 37 degrees C.
-Take supernatant to fresh tube.
-To count: Mix 25 ul Tc with 25 ul 16% PFA, mix, put 75 ul Cellometer cassette and count using the Cellometer Auto M10 (Nexcelom Biosciences).
-Wait for 2-5 min to let the Tc settle, press display image, focus, and count. Note, if there are numerous bubbles or low numbers of Tc, the counter will be inaccurate. It is critical to check every screen to see that it has properly counted. Often, there is ~ 10-20% over counting. Alternatively, TC can be counted a hemacytometer using the smaller/inner grids.
Leave Tc incubating in 50 ml conicals until ready to dilute for experiment or inoculate new LLC-MK2 culture.
-Aspirate LLC-MK2 media (used 10% for plating) and replace with 2%+10% Horse Serum FBS/DMEM.
-Plate 5-10 Million on Tc on fresh LLC-MK2
-24 hours later, wash cells with PBS and put fresh media on cells.without horse serum (Use 2% FBS/DMEM). If there are visibile Tc after 3 days, repeat PBS wash and changing of the media.-#Harvest Tc trypomastigotes on Day 7.

E. Growth inhibition assay for HTS in 384 well plates:
Notes:
-Final volume per well for cells+parasites+compound is 50 ul.
-Final volume after adding substrate is ~70 ul. (evaporation of 60ul to 40 ul after 90 hrs, and then 30ul GalScreen addition)
-The assay is performed in DMEM+ 2% FBS and 1% PSG .
-Experiment set up is usually done ~12pm. Assay has to be incubated 90 hrs or a bit more.
Protocol:
-warm up medium 2% FBS/DMEM
-harvest parasites in 50 ml tubes (1 flasks per tube)
-spin 10 min at 2200 rpm
-Aspirate media ~ 15 ml and place them on a rack in the incubator to let trypomastigotes swim out of the pellet for 3-5 hours.
-In the meantime, trypsinize NIH/3T3 cells as described in cell culture protocol
-When NIH3T3 are detached, harvest them in DMEM- 2% FBS and 1% PSG and count using the NIH3T3 program using Cellometer Auto T4 (Nexcelom Biosciences).
-Dilute cells to 166, 667 cells /ml and add to flask.
-plate 5,000 cells/30ul per well using a Thermo MultiDrop Combi liquid dispenser and a sterilized standard sized dispensing cassette adding at a fast speed in a tissue culture hood.
-Put back in incubator for 3 hours to allow cells to attach.
-Count Tc as from protocol above.
-Dilute to 166,667/ml and transfer a 2 liter flask.
-Pin 100 nl compounds/DMSO to each well
-Add shortly after 30 ul/well of parasites (5000 Tc) using a Thermo MultiDrop Combi liquid dispenser and a sterilized standard sized dispensing cassette adding at slow speed.
-Incubate for 4 days (or minimum 90 hours).
-On day of substrate addition, prepare GalScreen(Buffer B with 1:25 dilution substrate ).
-Add 30 ul per well of 384 well plate using a Thermo MultiDrop Combi liquid dispenser and a sterilized standard sized dispensing cassette adding at a fast speed.
-Incubate for 60 minutes
-Read using the ultrasensistive luminescence program on the Envision (Perkin Elmer, 0.1 sec/well).
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
All well activities were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention.


PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at -95.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 55.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: NIH 3T3
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1REPRODUCIBILITY_COSINE_TRANSFORMA measure of how well the activity reproduced across the two samples. Computed as the absolute value of the cosine between the 'replicate vector' (ScoreA, ScoreB ---as well as ScoreC and/or ScoreD where applicable) and the vector (1, 1) representing perfect reproducibility. NULL will appear in this column if a sample was not run in duplicate or if the data produced by one of the replicates was Invalid
Float
2PCT_ACTIVE_REPLICATESThe percentage of replicates which pass the activity threshold.Float
3REPLICATE_A_ACTIVITY_SCORE_8.35uM_(%) (8.35μM**)The calculated activity for the indicated sampleFloat%
4REPLICATE_B_ACTIVITY_SCORE_8.35uM_(%) (8.35μM**)The calculated activity for the indicated sampleFloat%

** Test 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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