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BioAssay: AID 651895

L-6 rat myocyte cell toxicity Measured in Cell-Based System Using Plate Reader - 2138-06_Inhibitor_Dose_DryPowder_Activity

This is a counter screen to look for host cell toxicity for T. cruzi experiments conducted in L6 cells. ..more
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 Tested Compounds
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Inactive(8)
 
 
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 Related BioAssays
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AID: 651895
Data Source: Broad Institute (2138-06_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 All Data
Tested Compounds:
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
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
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
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: L-6 myocyte, toxicity, host cell


Assay Overview:
This is a counter screen to look for host cell toxicity for T. cruzi experiments conducted in L6 cells.

Expected Outcome: Compounds significantly suppressing Alamar blue, and thus are toxic to L-6 myocytes, are considered active.
Protocol
In vitro cytotoxicity with L-6 cells. Assays were performed in 96-well microtiter plates, each well containing 100 ml of RPMI 1640 medium supplemented with 1% L-glutamine (200mM) and 10% fetal bovine serum, and 4000 L-6 cells (a primary cell line derived from rat skeletal myoblasts)( Page et al., 1993 and Ahmed et al., 1994). Serial drug dilutions of eleven 3-fold dilution steps covering a range from 100 to 0.002 ig/ml were prepared. After 70hours of incubation the plates were inspected under an inverted microscope to assure growth of the controls and sterile conditions. 10ml of Alamar Blue was then added to each well and the plates incubated for another 2 hours. 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). Podophyllotoxine is used as control.
C.Page C, M. Page and C. Noel. 1993. A new fluorimetric assay for cytotoxicity measurements in vitro.International Journal of Oncology 3: 473-476.
S.A. Ahmed, R.M. Gogal and J.E. Walsh. 1994. A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H] thymidine incorporation assay. The Journal of Immunological Methods 170: 211-224.
In vitro sensitivity assays: Cytotoxicity
Aug. 2007
Standard Cell Lines:
L-6 (rat skeletal myoblast cells)
Standard Drug:
Podophylotoxin (PPT); starting concentration: 0.1microg/ml
average IC50 = 0.006 microg/ml
Standard Conditions:
Medium: RPMI 1640 + 10% FCS + 1.7microM L-Glutamine (850microl 200mM for 100ml)
Culture vessel: CostarTM 96-well microtiter plates
Incubation: 37 degrees C, 5% CO2
Drug preparation:
Compounds are dissolved in DMSO (SOP Nr. D1), unless otherwise specified by the supplier. The stock solution is 10 mg/ml and stored at -20 degrees C. Stocks are kept for 3 years. (Since DMSO is toxic, care has to be taken not to exceed a final concentration of 1% DMSO in the assay).
Procedure:
1. Add 100microl of medium to wells of columns 3, 6, 9 and 12 of a microtiter plate. These wells serve as controls.
2. 100 microl of a cell suspension of 4x104 cells/ml is added into the remaining columns (1 and 2, 4 and 5, 7 and 8, and 10 and 11). Cells are allowed to attach over night. Per plate, allow for 6.5ml of cell suspension to be used.
3. The next day, the medium is removed from row H (do this for half of the plates and go to step 4 and return to step 3 for the second half, so the cells don't dry out).
4. 150 microl of medium containing the highest drug concentration is added to the wells of row H. Four drugs can be tested on one plate (drug 1 column 1-3, drug 2 column 4-6, etc.).
5. 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.
6. The plates are then incubated for 70 hrs at 37 masculineC / 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 the plates for another 2 hours (until a color change is observed, but maximum 3 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.
Data are transferred into a graphic program (Excel) and are evaluated to determine the IC50 or analyzed using the fluorescent plate reader software (SoftMax).
Comment
Compounds with IC50 less than 100 fold greater than the control IC50 (Podophyllotoxin) were labeled active.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Type: Toxicity
Assay Cell Type: L-6
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 All Data
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