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This page provides an example of the ISA-TAB-Nano Investigation File leveraging data from an Nanotechnology Characterization Laboratory (NCL) Investigation (NCL200612A). For the complete file, please refer the NCL Investigation File Example.

The ISA-TAB-Nano Investigation File consists of the following sections:

  • ONTOLOGY SOURCE REFERENCE
  • INVESTIGATION
  • INVESTIGATION PUBLICATIONS
  • INVESTIGATION CONTACTS
  • MATERIAL
  • STUDY
    • STUDY DESIGN DESCRIPTORS
    • STUDY PUBLICATIONS
    • STUDY FACTORS
    • STUDY ASSAYS
    • STUDY PROTOCOLS
    • STUDY CONTACTS

An example of each section is provided below. 

Ontology Source Reference

TABLE 1: Example Investigation File Ontology Source Reference Section

ABCDEFG
Term Source NameMONPOUOChEBIPATONCIt
Term Source Filehttp://purl.bioontology.org/ontology/MO Exit Disclaimer logo http://purl.bioontology.org/ontology/npo Exit Disclaimer logo http://purl.bioontology.org/ontology/UO Exit Disclaimer logo http://purl.bioontology.org/ontology/CHEBI Exit Disclaimer logo http://purl.bioontology.org/ontology/PATO Exit Disclaimer logo http://ncit.nci.nih.gov/
Term Source Versionv.1.3.1.1v.2011-02-12 v.80 v.11.11d
Term Source DescriptionMGED OntologyNanoParticle OntologyUnit OntologyChemical Entities of Biological InterestPhenotype OntologyNCI Thesaurus

Investigation

TABLE 2: Example Investigation File Investigation Section

AB
Investigation IdentifierNCL200612A
Investigation TitleDendrimer-Based MRI Contrast Agents
Investigation DescriptionThe goal of this investigation is to characterize a PAMAM dendrimer with an associated gadolinium chelate MRI contrast agent.
Investigation Submission Date2002-11-30
Investigation Public Release Date2002-11-30
Investigation Disease 
Investigation Disease Term Accession Number 
Investigation Disease Term Source REF 
Investigation Outcome 

Investigation Publications

TABLE 3. Example Investigation Publication Section

AB
Investigation PubMed ID18095846
Investigation Publication DOI10.2217/17435889.2.6.789  
Investigation Publication Author ListHall JB; Dobrovolskaia MA; Patri AK; McNeil SE
Investigation Publication TitleCharacterization of nanoparticles for therapeutics
Investigation Publication Statuspublished
Investigation Publication Status Term Accession Number 
Investigation Publication Status Term Source REF 

Investigation Contacts

TABLE 4: Example Investigation File Investigation Contacts Section

AB
Investigation Person Last NameDoe
Investigation Person First NameJohn
Investigation Person Mid InitialsE
Investigation Person Emaildoej@mail.nih.gov
Investigation Person Phone1231231234
Investigation Person Fax 
Investigation Person AddressLaboratory Street,  City,  State 111111
Investigation Person AffiliationDoe Laboratories
Investigation Person Rolesinvestigator
Investigation Person Roles Term Accession Number 
Investigation Person Roles Term Source REFMO

Material

TABLE 5. Example Investigation File Material Section

AB
Material File Namem_NCL-21.xls
Material Source NameNCL-21

Study

TABLE 6. Example Investigation File Study Section

AB
Study IdentifierNCL200612A-CytoxicityLLC-PK1
Study TitleCytotoxicity characterization in LLC-PK1 cells
Study DescriptionNanoparticle biocompatibility was evaluated in the porcine renal proximal tubule cell   line, LLC-PK1. Cytotoxicity was determined as described in the NCL protocol for LLC-PK1 Kidney Cytotoxicity Assay(GTA-1). Briefly, test materials were diluted to the desired assay concentrations in cell culture media. Cells were preincubated for 24 h prior to adding test material, reaching an approximate confluence of 80%. Cells were exposed to test material for 6, 24 and 48 h, and cytotoxicity was determined using the MTT cell viability and LDH membrane integrity assays.
Study Submission Date2002-11-30
Study Public Release Date2002-11-30
Study Disease 
Study Disease Term Accession Number 
Study Disease Term Source REF 
Study OutcomeNCL22, NCL23 and NCL24 were found to be minimally cytotoxic, under the testing   conditions utilized.
Study File Names_cytotoxicity-LLC-PK1.xls

Study Design Descriptors

TABLE 7. Example Investigation File Study Design Descriptors Section

AB
Study Design Typecomparison
Study Design Type Term Accession Number 
Study Design Type Term Source REF 

Study Publications

TABLE 8. Example Investigation File Study Publications Section

AB
Study PubMed ID18095846
Study Publication DOI10.2217/17435889.2.6.789  
Study Publication Author listHall JB; Dobrovolskaia MA; Patri AK; McNeil SE
Study Publication TitleCharacterization of nanoparticles for therapeutics
Study Publication Statuspublished
Study Publication Status Term Accession Number 
Study Publication Status Term Source REF 

Study Factors

TABLE 9a. Example Investigation File Study Factors Section for Study Involving Biospecimens (e.g. in vitro, in vivo characterization). The study factor name and type must be of nanoparticle sample if the assay is applying a nanoparticle sample to a biological system.

ABCD
Study Factor Namenanoparticle sampledosetime of exposure
Study Factor Typenanoparticle sampleparticle concentrationtime of exposure
Study Factor Type Term Accession NumberNPO_1404NPO_1830NPO_1819
Study Factor Type Term Source REFNPONPONPO


TABLE 9b. Example Investigation File Study Factors Section for Physico-Chemical Characterization Study. There should be no study factors of study factor type nanoparticle sample 

ABC
Study Factor Nametemperaturesolvent
Study Factor Typetemperaturesolvent medium
Study Factor Type Term Accession NumberPATO_0000146NPO_1855
Study Factor Type Term Source REFPATONPO

Study Assays

TABLE 10. Example Investigation File Study Assays Section

ABC
Study Assay Measurement TypeMTT AssayLDH Release Assay
Study Assay Measurement Type Term Accession Number NPO_1709
Study Assay Measurement Type Term Source REF NPO
Study Assay Technology Type  
Study Assay Technology Type Term Accession Number  
Study Assay Technology Type Term Source REF  
Study Assay Technology Platform  
Study Assay Measurement Namecell viabilityLDH release
Study Assay Measurement Name Term Accession NumberNPO_1343 
Study Assay Measurement Name Term Source REFNPO 
Study Assay File Namea_MTT-LLCPK1.xlsa_LDH-LLCPK1.xls

Study Protocols

TABLE 11. Example Investigation File Study Protocols Section

ABCDEFGHIJ
Study Protocol   NameTime-6-24-48 plate   MTT assayTest plate LDH assayTime zero plate MTT   assaycell preparation in four 96-well platesAPAP positive control preparationTriton-X100 positive control preparationMTT assay reagent preparationTest sample and   positive control additionLDH assay reagent preparation
Study   Protocol Type   cell preparationcontrol preparationcontrol preparationreagent preparation reagent preparation
Study   Protocol Type Term Accession Number         
Study   Protocol Type Term Source REF         
Study   Protocol DescriptionTest   Plates: 6, 24 and 48 hour exposures (MTT Assay) 5.4.1 Remove appropriate test plate from incubator and replace media from   Triton-X positive control wells (see plate format in Appendix) with 200 mL 1% Triton-X (made in Step 4.1.2).   Let the plate set for 10 minutes at room temperature. Spin plate at 700 x g   for 3 minutes.
    5.4.2 Remove 100 mL   of media from each well and transfer it to another plate, maintaining plate   format. Use this plate immediately for the LDH assay (see Section 5.5).
    5.4.3 Remove remaining media from original plate and discard.
    5.4.4 Add 200 mL of   fresh media to all wells.
    5.4.5 Add 50 mL of   MTT to all wells.
    5.4.6 Cover in aluminum foil and incubate for 37°C for 4 hours.
    5.4.7 Remove plate from incubator and spin at 700 x g for 3 minutes.
    5.4.8 Remove media and MTT.
    5.4.9 Add 200 mL of   DMSO to each well.
    5.4.10 Add 25 mL of   glycine buffer to each well. Place on shaker to mix.
    5.4.11 Read absorbance at 570 nm on plate reader using a reference   wavelength of 680 nm.
Test   Plates: 0, 6, 24 and 48 hour exposures (LDH Assay) Adapted from Biovision LDH Cytotoxicity Assay Kit, K311-400)
    5.5.1 Add 100 mL of the   Reaction Mixture (step 4.3.2) to each well of transfer plate. Shake plate on   an orbital shaker briefly to mix samples.
    5.5.2 Incubate at room temperature for up to 20 minutes in the dark.
    5.5.3 Read the plate on plate reader at 490 nm using a reference wavelength   of 680 nm.
5.2.1   Remove time zero plate from incubator and replace media from Triton-X positive control wells (see plate format in Appendix) with 200 mL 1% Triton-X (made in Step 4.1.2).   Add 100 mL of media to   the remaining wells. Let the plate set for 10 minutes at room temperature.   Spin plate at 700 x g for 3 minutes.
    5.2.2 Remove 100 mL   of media from each well and transfer it to another plate, maintaining plate   format. Use this plate immediately for the LDH assay (see Section 5.5).
5.2.3 Remove remaining media from original plate and discard.
5.2.4 Add 200 mL of  fresh media to all wells. 5.2.5 Add 50 mL of   MTT to all wells. 5.2.6 Cover in aluminum foil and incubate at 37°C for 4 hours. 5.2.7 Remove plate from incubator and spin at 700 x g for 3 minutes. 5.2.8 Aspirate media and MTT. 5.2.9 Add 200 mL of   DMSO to all wells. 5.2.10 Add 25 mL of   glycine buffer to all wells. Place on shaker to mix. 5.2.11 Read absorbance at 570 nm on plate reader.
5.1.1   Harvest cryopreserved cells from prepared flasks (limit to 20   passages). 5.1.2 Count cell concentration using a coulter counter or  hemocytometer.
  5.1.3 Dilute cells to a density of 2.5 x 105 cells/mL in M199 (3% FBS) cell   culture media.
    5.1.4 Plate 100 mL   cells/well as per plate format (Appendix) for four 96-well plates (time zero,   6 hour sample exposure, 24 hour sample exposure, 48 hour sample exposure).   The format indicates no cells in rows D and E as they serve as particle   blanks to be subtracted from cell treatment wells. Each plate accommodates   two samples (Rows A–C and F–H). Each nanoparticle is tested at nine   dilutions. Column 11 receives the APAP positive control and column 12   receives Triton X-100. 5.1.5 Incubate plates for 24 hours at 5% CO2, 37°C and 95% humidity.
4.1.1   Acetaminophen (APAP) positive control: Add 19 mg to a total volume of 5 mL   M199 Cell Culture Media (with 3% FBS) to make a 25 mM solution. Sterile   filter using a 0.2 mm filter.4.1.2   1% Triton-X-100 positive control: Add 1 mL of Triton-X-100 to 99 mL of media.   Sterile filter using a 0.2 mm   filter.4.2.1   MTT solution: 5 mg/mL MTT in PBS, store for up to one month at 4°C in dark 4.2.2 Glycine Buffer: 0.1 M glycine (MW 75.07), 0.1 M NaCl (MW   58.44), pH 10.5, store at room temperature.5.3.1   The highest concentration of nanoparticle tested should be at the limit of solubility. The test sample should be at physiological pH. Neutralization of   acidic/basic test samples may be required. 5.3.2 Dilute test compound in media, making a total of 9 1:4 dilutions. 5.3.3 Add 100 mL of each   sample dilution and positive control to 6 hour, 24 hour and 48 hour exposure   plates as per the plate format (Appendix).4.3.1   Reconstitute catalyst in 1 mL dH20 for 10 min with occasional vortexing   (stable for 2 weeks at 4°C). 4.3.2 Reaction mixture (for one 96-well plate): Add 250 mL of reconstituted catalyst solution to 11.25 mL of dye   solution (stable for 2 weeks at 4°C).
Study   Protocol URINCL_Method_GTA-1.pdfNCL_Method_GTA-1.pdfNCL_Method_GTA-1.pdfNCL_Method_GTA-1.pdfNCL_Method_GTA-1.pdfNCL_Method_GTA-1.pdfNCL_Method_GTA-1.pdfNCL_Method_GTA-1.pdfNCL_Method_GTA-1.pdf
Study   Protocol Version1.11.11.11.11.11.11.11.11.1
Study   Protocol Parameters Name         
Study   Protocol Parameters Name Term Accession Number         
Study   Protocol Parameters Name Term Source REF         
Study   Protocol Components NameMTT;   acetaminophen; dimethyl sulfoxide; glycine; sodium chloride; triton-X-100;   M199 cell culture media; fetal bovine serum; nanoparticle; costar 96 well   flat bottom cell culture plates; plate reader; centrifuge set at 700-800 x g   (Allegra X-15R, Beckman Coulter) with 96 well plate adapter; orbital plate   shakeracetaminophen;   dimethyl sulfoxide; glycine; sodium chloride; triton-X-100; M199 cell culture   media; fetal bovine serum; biovision LDH-cytoxicity assay kit; nanoparticle;   costar 96 well flat bottom cell culture plates; plate reader; centrifuge set   at 700-800 x g (Allegra X-15R, Beckman Coulter) with 96 well plate adapter;   orbital plate shakerMTT;   acetaminophen; dimethyl sulfoxide; glycine; sodium chloride; triton-X-100;   M199 cell culture media; fetal bovine serum; costar 96 well flat bottom cell   culture plates; plate reader; centrifuge set at 700-800 x g (Allegra X-15R,   Beckman Coulter) with 96 well plate adapter; orbital plate shakerM199   cell culture media; fetal bovine serum; costar 96 well flat bottom cell   culture plates; plate reader; centrifuge set at 700-800 x g (Allegra X-15R,   Beckman Coulter) with 96 well plate adapter; orbital plate shakeracetaminophen;   M199 cell culture media; fetal bovine serumtriton-X-100;   M199 cell culture media; fetal bovine serumMTT;glycine;   sodium chlorideacetaminophen;glycine;   sodium chloride; triton-X-100; M199 cell culture media; fetal bovine serum;   nanoparticle; costar 96 well flat bottom cell culture plates; plate reader;   centrifuge set at 700-800 x g (Allegra X-15R, Beckman Coulter) with 96 well   plate adapter; orbital plate shakerbiovision   LDH-cytoxicity assay kit
Study Protocol   Components Typereagent;   reagent; reagent; reagent; reagent; reagent; reagent; reagent; reagent;   material; instrument; instrument; instrumentreagent;   reagent; reagent; reagent; reagent; reagent; reagent; reagent; reagent;   material; instrument; instrument; instrumentreagent;   reagent; reagent; reagent; reagent;reagent; reagent; reagent; material;   instrument; instrument; instrumentreagent;   reagent;material; instrument; instrument; instrumentreagent;   reagent; reagentreagent;   reagent; reagentreagent;   reagent; reagentreagent;   reagent; reagent; reagent; reagent; reagent; reagent; material; instrument;   instrument; instrumentreagent
Study Protocol   Components Type Term Accession NumberNPO_290;   NPO_290; NPO_290; NPO_290; NPO_290; NPO_290; NPO_290; NPO_290;NPO_290; ;   NPO_1436; NPO_1436; NPO_1436NPO_290;   NPO_290; NPO_290; NPO_290; NPO_290; NPO_290; NPO_290; NPO_290;NPO_290; ;   NPO_1436; NPO_1436; NPO_1436NPO_290; NPO_290; NPO_290;   NPO_290; NPO_290; NPO_290; NPO_290;NPO_290; ; NPO_1436; NPO_1436; NPO_1436NPO_290; NPO_290; ; NPO_1436;   NPO_1436; NPO_1436NPO_290; NPO_290; NPO_290NPO_290;   NPO_290; NPO_290NPO_290;   NPO_290; NPO_290NPO_290; NPO_290; NPO_290;   NPO_290; NPO_290; NPO_290; NPO_290; ; NPO_1436; NPO_1436; NPO_1436NPO_290
Study Protocol   Components Type Term Source REFNPO;   NPO; NPO; NPO; NPO; NPO; NPO; NPO; NPO; ; NPO; NPO; NPONPO;   NPO; NPO; NPO; NPO; NPO; NPO; NPO; NPO; ; NPO; NPO; NPONPO; NPO; NPO;   NPO; NPO; NPO; NPO; NPO; ; NPO; NPO; NPONPO; NPO; ;   NPO; NPO; NPONPO; NPO; NPONPO;   NPO; NPONPO;   NPO; NPONPO; NPO; NPO;   NPO; NPO; NPO; NPO;  ; NPO; NPO; NPONPO

Study Contacts

TABLE 12. Example Investigation File Study Contacts Section

AB
Study Person Last NameSmith
Study Person First NameJane
Study Person Mid InitialsK
Study Person Emailsmithj@mail.nih.gov
Study Person Phone1231231235
Study Person Fax 
Study Person AddressLaboratory Street,  City,  State 111111
Study Person AffiliationDoe Laboratories
Study Person Rolesinvestigator
Study Person Roles Term Accession Number 
Study Person Roles Term Source REFMO
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