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BioAssay: AID 493197

Intracellular Trypanosomes Measured in Cell-Based/Microorganism System Using Plate Reader - 2017-01_Inhibitor_Dose_DryPowder_Activity_Set3

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(46)
 
 
Active(27)
 
 
Inactive(12)
 
 
Inconclusive(7)
 
 
 Tested Substances
 Tested Substances
All(46)
 
 
Active(27)
 
 
Inactive(12)
 
 
Inconclusive(7)
 
 
 Related BioAssays
 Related BioAssays
AID: 493197
Data Source: Broad Institute (2017-01_Inhibitor_Dose_DryPowder_Activity_Set3)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-02-10
Hold-until Date: 2011-03-02
Modify Date: 2011-03-02

Data Table ( Complete ):           Active    All
BioActive Compounds: 27
Depositor Specified Assays
AIDNameTypeComment
1968Broad Institute MLPCN T. Cruzi Inhibition ProjectsummarySummary 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)
-NIH/3T3 cells (mouse embryonic fibroblast cell line)
-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.)
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
-NP40 (Nonidet P-40, now called Igepal CA 360, Fluka, cat number: 56741)
-GalScreen, Buffer B (Applied Biosystems, cat number: T1031)
Materials:
-T175, 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, 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 + 0.05% NP40. Using GalScreen base kit (Applied Biosystems), mix Buffer B (Applied Biosystems, T2361) with 1:25 substrate (Applied Biosystems, T2359) with 1:400 dilution of 20%NP40.

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 T175 or Corning Hyperflasks .
-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-a-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 3 Million LLC-MK2 cells/T175 in DMEM+10% 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 3 hours at 37 degrees C.
-Take supernatant to fresh tube.
-To count: Mix 75 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% FBS/DMEM.
-Plate 17-35 Million on Tc on fresh LLC-MK2
-Change medium after 2 days (Use 2% FBS/DMEM).
-Harvest Tc trypomastigotes on Day 5, 6 and/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 ~65 ml. (evaporation of 50ul to 35ul 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 0.250 million /ml and transfer a 2 liter flask.
-Pin 50 nl compounds/DMSO to each well
-Add shortly after 20 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 with 0.05% NP40 (Buffer B with 1:25 dilution substrate with 1:400 dilution of 20% NP40).
-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).
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=104) and positive control wells (PC; n=20) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (10)(Max_Concentration).

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(NC).
A normalized activity value of -50 is defined as (0.5)(NC).
Experimental wells values were scaled to this range.

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

MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)

PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.

PUBCHEM_ACTIVITY_SCORE:
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.

Note:
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_Qualifier'>','=', or '<'String
2AC50_uM*The concentration at which activity reaches 50% of the maximumFloatμM
3pAC50_MEqual to -1*log10(AC50)String
4PUBCHEM_ACTIVITY_OUTCOME_TEST1Integer
5PUBCHEM_ACTIVITY_SCORE_TEST1The Activity Summary for every Substance has two parts, the outcome and the score. The score for this Substance for this test is reported in this column and must be an integer value, where larger values are more active and smaller values are less active. Please make sure your scores are on a linear scale because that's how they will be interpreted. We encourage depositors to consider using the range 0-100, although values larger and smaller are allowed. The score values are used to allow PubChem users to partition, sort, and profile Assay Data results within and between biological assays.Integer
6PUBCHEM_ASSAYDATA_COMMENT_TEST1The Activity Summary for every Substance has two parts, the outcome and the score. The outcome for each Substance for this test is reported as an integer value in this column and must be one of three different values:
1 - Substance is considered inactive
2 - Substance is considered active
3 - Substance activity outcome is inconclusive
5 - Substance is considered probe
Integer
7AC50_Qualifier_TEST1'>','=', or '<'String
8AC50_uM_TEST1The concentration at which activity reaches 50% of the maximumFloatμM
9pAC50_M_TEST1Equal to -1*log10(AC50)String
10Hill_Slope_TEST1The slope at AC50Float
11S0_(%)_TEST1The fitted activity value at zero concentrationFloat%
12Sinf_(%)_TEST1The fitted activity value at infinite concentrationFloat%
13Num_Points_TEST1The number of data points used to generate the plotInteger
14Max_Activity_(%)_TEST1The maximum activity value observed, based on mean of replicates per concentrationFloat%
15Max_Activity_Conc_uM_TEST1The concentration at which the maximum activity is observedFloatμM
16Max_Concentration_uM_TEST1Maximum valid test concentrationFloatμM
17Activity_at_0.003uM_(%)_TEST1 (0.003μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_0.009uM_(%)_TEST1 (0.009μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_0.0285uM_(%)_TEST1 (0.0285μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_0.08uM_(%)_TEST1 (0.08μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_0.235uM_(%)_TEST1 (0.235μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_0.75uM_(%)_TEST1 (0.75μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_2.1uM_(%)_TEST1 (2.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_5uM_(%)_TEST1 (5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25Activity_at_6.8uM_(%)_TEST1 (6.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
26Activity_at_19.5uM_(%)_TEST1 (19.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
27PUBCHEM_ACTIVITY_OUTCOME_TEST2The Activity Summary for every Substance has two parts, the outcome and the score. The outcome for each Substance for this test is reported as an integer value in this column and must be one of three different values:
1 - Substance is considered inactive
2 - Substance is considered active
3 - Substance activity outcome is inconclusive
5 - Substance is considered probe
Integer
28PUBCHEM_ACTIVITY_SCORE_TEST2The Activity Summary for every Substance has two parts, the outcome and the score. The score for this Substance for this test is reported in this column and must be an integer value, where larger values are more active and smaller values are less active. Please make sure your scores are on a linear scale because that's how they will be interpreted. We encourage depositors to consider using the range 0-100, although values larger and smaller are allowed. The score values are used to allow PubChem users to partition, sort, and profile Assay Data results within and between biological assays.Integer
29PUBCHEM_ASSAYDATA_COMMENT_TEST2String
30AC50_Qualifier_TEST2'>','=', or '<'String
31AC50_uM_TEST2The concentration at which activity reaches 50% of the maximumFloatμM
32pAC50_M_TEST2Equal to -1*log10(AC50)String
33Hill_Slope_TEST2The slope at AC50Float
34S0_(%)_TEST2The fitted activity value at zero concentrationFloat%
35Sinf_(%)_TEST2The fitted activity value at infinite concentrationFloat%
36Num_Points_TEST2The number of data points used to generate the plotInteger
37Max_Activity_(%)_TEST2The maximum activity value observed, based on mean of replicates per concentrationFloat%
38Max_Activity_Conc_uM_TEST2The concentration at which the maximum activity is observedFloatμM
39Max_Concentration_uM_TEST2Maximum valid test concentrationFloatμM
40Activity_at_0.003uM_(%)_TEST2 (0.003μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
41Activity_at_0.009uM_(%)_TEST2 (0.009μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
42Activity_at_0.0285uM_(%)_TEST2 (0.0285μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
43Activity_at_0.08uM_(%)_TEST2 (0.08μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
44Activity_at_0.235uM_(%)_TEST2 (0.235μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
45Activity_at_0.75uM_(%)_TEST2 (0.75μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
46Activity_at_2.1uM_(%)_TEST2 (2.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
47Activity_at_5uM_(%)_TEST2 (5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
48Activity_at_6.8uM_(%)_TEST2 (6.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
49Activity_at_19.5uM_(%)_TEST2 (19.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
50PUBCHEM_ACTIVITY_OUTCOME_TEST3The Activity Summary for every Substance has two parts, the outcome and the score. The outcome for each Substance for this test is reported as an integer value in this column and must be one of three different values:
1 - Substance is considered inactive
2 - Substance is considered active
3 - Substance activity outcome is inconclusive
5 - Substance is considered probe
Integer
51PUBCHEM_ACTIVITY_SCORE_TEST3The Activity Summary for every Substance has two parts, the outcome and the score. The score for this Substance for this test is reported in this column and must be an integer value, where larger values are more active and smaller values are less active. Please make sure your scores are on a linear scale because that's how they will be interpreted. We encourage depositors to consider using the range 0-100, although values larger and smaller are allowed. The score values are used to allow PubChem users to partition, sort, and profile Assay Data results within and between biological assays.Integer
52PUBCHEM_ASSAYDATA_COMMENT_TEST3String
53AC50_Qualifier_TEST3'>','=', or '<'String
54AC50_uM_TEST3The concentration at which activity reaches 50% of the maximumFloatμM
55pAC50_M_TEST3Equal to -1*log10(AC50)String
56Hill_Slope_TEST3The slope at AC50Float
57S0_(%)_TEST3The fitted activity value at zero concentrationFloat%
58Sinf_(%)_TEST3The fitted activity value at infinite concentrationFloat%
59Num_Points_TEST3The number of data points used to generate the plotInteger
60Max_Activity_(%)_TEST3The maximum activity value observed, based on mean of replicates per concentrationFloat%
61Max_Activity_Conc_uM_TEST3The concentration at which the maximum activity is observedFloatμM
62Max_Concentration_uM_TEST3Maximum valid test concentrationFloatμM
63Activity_at_0.003uM_(%)_TEST3 (0.003μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
64Activity_at_0.009uM_(%)_TEST3 (0.009μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
65Activity_at_0.0285uM_(%)_TEST3 (0.0285μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
66Activity_at_0.08uM_(%)_TEST3 (0.08μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
67Activity_at_0.235uM_(%)_TEST3 (0.235μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
68Activity_at_0.75uM_(%)_TEST3 (0.75μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
69Activity_at_2.1uM_(%)_TEST3 (2.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
70Activity_at_5uM_(%)_TEST3 (5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
71Activity_at_6.8uM_(%)_TEST3 (6.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
72Activity_at_19.5uM_(%)_TEST3 (19.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

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

Data Table (Concise)
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