Example Files:
This page provides examples for the ISA-TAB-Nano Investigation files, leveraging data from the Nanotechnology Characterization Laboratory (NCL) as an example (NCL200612A).
TABLE 1. ISA-TAB-Nano Investigation File Extensions to ISA-TAB
Section | Field | Field Status (if applicable) |
|---|---|---|
INVESTIGATION | Investigation disease | Required |
INVESTIGATION | Investigation disease term accession number | Required |
INVESTIGATION | Investigation disease term source REF | Required |
INVESTIGATION | Investigation outcome | Optional |
STUDY | Study disease | Required |
STUDY | Study disease term accession number | Required |
STUDY | Study disease term source REF | Required |
STUDY | Study outcome | Optional |
STUDY ASSAYS | Study assay measurement name | Required |
STUDY ASSAYS | Study assay measurement name term accession number | Required |
STUDY ASSAYS | Study assay measurement name term source REF | Required |
Ontology Source Reference
TABLE 2: Example Investigation File Ontology Source Reference Section
| ONTOLOGY SOURCE REFERENCE | ||||||
| Term Source Name | MO | NPO | UO | ChEBI | PATO | NCIt |
| Term Source File | http://purl.bioontology.org/ontology/MO | http://purl.bioontology.org/ontology/npo | http://purl.bioontology.org/ontology/UO | http://purl.bioontology.org/ontology/CHEBI | http://purl.bioontology.org/ontology/PATO | http://ncit.nci.nih.gov/ |
| Term Source Version | v. 1.3.1.1 | v. 2011-02-12 | v. 80 | v. 11.11d | ||
| Term Source Description | MGED Ontology | NanoParticle Ontology | Unit Ontology | Chemical Entities of Biological Interest | Phenotype Ontology | NCI Thesaurus |
Investigation
TABLE 3: Example Investigation File Investigation Section
| INVESTIGATION | |
| Investigation Identifier | NCL200612A |
| Investigation Title | Dendrimer-Based MRI Contrast Agents |
| Investigation Description | The goal of this investigation is to characterize a PAMAM dendrimer with an associated gadolinium chelate MRI contrast agent. |
| Investigation Disease | |
| Investigation Disease Term Accession Number | |
| Investigation Disease Term Source REF | |
| Investigation Outcome | |
| Investigation Submission Date | 2002-11-30 |
| Investigation Public Release Date | 2002-11-30 |
Investigation Contacts
TABLE 4: Example Investigation File Investigation Contacts Section
| INVESTIGATION CONTACTS | |
| Investigation Person Last Name | McNeil |
| Investigation Person First Name | Scott |
| Investigation Person Mid Initials | E |
| Investigation Person Email | mcneils@mail.nih.gov |
| Investigation Person Phone | 3018466939 |
| Investigation Person Fax | |
| Investigation Person Address | MSC 1050 Boyles Street, Frederick, MD 21702 |
| Investigation Person Affiliation | Nanotechnology Characterization Laboratory |
| Investigation Person Roles | investigator |
| Investigation Person Roles Term Accession Number | |
| Investigation Person Roles Term Source REF | MO |
Investigation Publication
TABLE 5. Example Investigation Publication Section
| INVESTIGATION PUBLICATIONS | |
| Investigation PubMed ID | 18095846 |
| Investigation Publication DOI | 10.2217/17435889.2.6.789 |
| Investigation Publication Author list | Hall JB; Dobrovolskaia MA; Patri AK; McNeil SE |
| Investigation Publication Title | Characterization of nanoparticles for therapeutics |
| Investigation Publication Status | published |
| Investigation Publication Status Term Accession Number | |
| Investigation Publication Status Term Source REF |
Study
TABLE 6. Example Investigation File Study Section
| STUDY | |
| Study Identifier | NCL200612A-CytoxicityLLC-PK1 |
| Study Title | Cytotoxicity characterization in LLC-PK1 cells |
| Study Submission Date | 2002-11-30 |
| Study Public Release Date | 2002-11-30 |
| Study Description | Nanoparticle 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 Disease | |
| Study Disease Term Accession Number | |
| Study Disease Term Source REF | |
| Study Outcome | NCL22, NCL23 and NCL24 were found to be minimally cytotoxic, under the testing conditions utilized. |
| Study File Name | s_cytotoxicity-LLC-PK1.xls |
Study Design Descriptors
TABLE 7. Example Investigation File Study Design Descriptors Section
| STUDY DESIGN DESCRIPTORS | |
| Study Design Type | comparison |
| Study Design Type Term Accession Number | |
| Study Design Type Term Source REF |
Study Contacts
TABLE 8. Example Investigation File Study Contacts Section
| STUDY CONTACTS | |
| Study Person Last Name | Dobrovolskaia |
| Study Person First Name | Marina |
| Study Person Mid Initials | A |
| Study Person Email | marina@mail.nih.gov |
| Study Person Phone | 3018466352 |
| Study Person Fax | |
| Study Person Address | MSC 1050 Boyles Street, Frederick, MD 21702 |
| Study Person Affiliation | Nanotechnology Characterization Laboratory |
| Study Person Roles | investigator |
| Study Person Roles Term Accession Number | |
| Study Person Roles Term Source REF | MO |
Study Publications
TABLE 9. Example Investigation File Study Publications Section
| STUDY PUBLICATIONS | |
| Study PubMed ID | 18095846 |
| Study Publication DOI | 10.2217/17435889.2.6.789 |
| Study Publication Author list | Hall JB; Dobrovolskaia MA; Patri AK; McNeil SE |
| Study Publication Title | Characterization of nanoparticles for therapeutics |
| Study Publication Status | published |
| Study Publication Status Term Accession Number | |
| Study Publication Status Term Source REF |
Study Assays
TABLE 10. Example Investigation File Study Assays Section
| STUDY ASSAYS | ||
| Study Assay Measurement Type | MTT Assay | LDH 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 Name | cell viability | LDH release |
| Study Assay Measurement Name Term Accession Number | NPO_1343 | |
| Study Assay Measurement Name Term Source REF | NPO | |
| Study Assay File Name | a_MTT-LLCPK1.xls | a_LDH-LLCPK1.xls |
Study Factors
TABLE 11. Example Investigation File Study Factors Section
| STUDY FACTORS | |||
| Study Factor Name | nanoparticle sample | particle concentration | time of exposure |
| Study Factor Name Term Accession Number | NPO_1404 | NPO_1830 | NPO_1819 |
| Study Factor Name Term Source REF | NPO | NPO | NPO |
| Study Factor Type | |||
| Study Factor Type Term Accession Number | |||
| Study Factor Type Term Source REF |
Study Protocols
TABLE 12. Example Investigation File Study Protocols Section
| STUDY PROTOCOLS | |||||||||
| Study Protocol Name | Time-6-24-48 plate MTT assay | Test plate LDH assay | Time zero plate MTT assay | cell preparation in four 96-well plates | APAP positive control preparation | Triton-X-100 positive control preparation | MTT assay reagent preparation | Test sample and positive control addition | LDH assay reagent preparation |
| Study Protocol Type | cell preparation | control preparation | control preparation | reagent preparation | reagent preparation | ||||
| Study Protocol Type Term Accession Number | |||||||||
| Study Protocol Type Term Source REF | |||||||||
| Study Protocol Description | Test 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 URI | NCL_Method_GTA-1.pdf | NCL_Method_GTA-1.pdf | NCL_Method_GTA-1.pdf | NCL_Method_GTA-1.pdf | NCL_Method_GTA-1.pdf | NCL_Method_GTA-1.pdf | NCL_Method_GTA-1.pdf | NCL_Method_GTA-1.pdf | NCL_Method_GTA-1.pdf |
| Study Protocol Version | 1.1 | 1.1 | 1.1 | 1.1 | 1.1 | 1.1 | 1.1 | 1.1 | 1.1 |
| Study Protocol Parameters Name | |||||||||
| Study Protocol Parameters Name Term Accession Number | |||||||||
| Study Protocol Parameters Name Term Source REF | |||||||||
| Study Protocol Components Name | MTT; 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 shaker | acetaminophen; 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 shaker | MTT; 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 shaker | 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 shaker | acetaminophen; M199 cell culture media; fetal bovine serum | triton-X-100; M199 cell culture media; fetal bovine serum | MTT;glycine; sodium chloride | acetaminophen;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 shaker | biovision LDH-cytoxicity assay kit |
| Study Protocol Components Type | reagent; reagent; reagent; reagent; reagent; reagent; reagent; reagent; reagent; material; instrument; instrument; instrument | reagent; reagent; reagent; reagent; reagent; reagent; reagent; reagent; reagent; material; instrument; instrument; instrument | reagent; reagent; reagent; reagent; reagent;reagent; reagent; reagent; material; instrument; instrument; instrument | reagent; reagent;material; instrument; instrument; instrument | reagent; reagent; reagent | reagent; reagent; reagent | reagent; reagent; reagent | reagent; reagent; reagent; reagent; reagent; reagent; reagent; material; instrument; instrument; instrument | reagent |
| Study Protocol Components Type Term Accession Number | NPO_290; NPO_290; NPO_290; NPO_290; NPO_290; NPO_290; NPO_290; NPO_290;NPO_290; ; NPO_1436; NPO_1436; NPO_1436 | NPO_290; NPO_290; NPO_290; NPO_290; NPO_290; NPO_290; NPO_290; NPO_290;NPO_290; ; NPO_1436; NPO_1436; NPO_1436 | NPO_290; NPO_290; NPO_290; NPO_290; NPO_290; NPO_290; NPO_290;NPO_290; ; NPO_1436; NPO_1436; NPO_1436 | NPO_290; NPO_290; ; NPO_1436; NPO_1436; NPO_1436 | NPO_290; NPO_290; NPO_290 | NPO_290; NPO_290; NPO_290 | NPO_290; NPO_290; NPO_290 | NPO_290; NPO_290; NPO_290; NPO_290; NPO_290; NPO_290; NPO_290; ; NPO_1436; NPO_1436; NPO_1436 | NPO_290 |
| Study Protocol Components Type Term Source REF | NPO; NPO; NPO; NPO; NPO; NPO; NPO; NPO; NPO; ; NPO; NPO; NPO | NPO; NPO; NPO; NPO; NPO; NPO; NPO; NPO; NPO; ; NPO; NPO; NPO | NPO; NPO; NPO; NPO; NPO; NPO; NPO; NPO; ; NPO; NPO; NPO | NPO; NPO; ; NPO; NPO; NPO | NPO; NPO; NPO | NPO; NPO; NPO | NPO; NPO; NPO | NPO; NPO; NPO; NPO; NPO; NPO; NPO; ; NPO; NPO; NPO | NPO |
