The Nanotechnology Characterization Laboratory (NCL) has developed a standardized analytical assay cascade to assist nanotechnology-based drug and device developers with preclinical characterization aimed at defining nanoparticle physicochemical properties, immunological compatibility, general toxicity, and pharmacology. Such characterization is essential for the translation of nanotechnology formulations from the discovery phase into clinical trials. The protocols were developed in close collaboration with the US Food and Drug Administration (US FDA) and the National Institute of Standards and Technology (NIST) and used since 2005 to characterize over a thousand nanoformulations, including but not limited to lipid nanoparticles, liposomes, polymeric micelles, metal colloids and metal oxides, carbon nanotubes, fullerenes, dendrimers, and nucleic acid nanoparticles. The protocols can also be used for characterization of nanomaterials for other types of non-clinical studies.

Contents

• Immunology protocols
  • NCL Method ITA-37.2. Immunophenotyping: Analysis of Nanoparticle Effects on the Composition and Activation Status of Human Peripheral Blood Mononuclear Cells: Version 3
    Hannah S. Newton, Jenny Zhang, and Marina A. Dobrovolskaia.
    Published: November 2023.
    • 1. Introduction
    • 2. Principle
    • 3. Reagents, Materials, and Equipment
    • 4. Preparation of Reagents and Controls
    • 5. Preparation of Nanoparticles
    • 6. NovoCyte 3005 Instrument Settings
    • 7. Experimental Conditions Necessary for Preparation of Immunophenotyping Panels #1 and #2
    • 8. Collection and Handling of Whole Blood (Day 1)
    • 9. Preparation of PBMC
    • 10. Experimental Procedure for Nanomaterial Treatment of PBMC (24-well)
    • 11. Experimental PBMC Staining Procedure (Day 2)
    • 12. Data Acquisition on Novocyte 3005 (Day 3)
    • 13. Data Analysis of Immunophenotyping Panels with FCS Express
    • 14. References
    • 15. Abbreviations
  • NCL Method ITA-37.1. Immunophenotyping: Instrument Calibration and Reagent Qualification for Immunophenotyping Analysis of Human Peripheral Blood Mononuclear Cell Cultures: Version 3
    Hannah S. Newton, Jenny Zhang, and Marina A. Dobrovolskaia.
    Published: November 2023.
    • 1. Introduction
    • 2. Principle
    • 3. Reagents, Materials, and Equipment
    • 4. Preparation of Reagents and Controls
    • 5. NovoCyte 3005 Instrument Settings
    • 6. Flow Cytometry Control Experiments with Compensation Beads for Antibody Titration and Single Stain Controls
    • 7. Flow Cytometry Control Experiments with PBMC (FMO Controls) (2 Day procedure per panel)
    • 8. References
    • 9. Abbreviations
    • 10. Appendix
  • NCL Method ITA-29. Detection of nanoparticles’ ability to stimulate toll-like receptors using HEK-Blue reporter cell lines: Version 2
    Edward Cedrone and Marina A. Dobrovolskaia.
    Published: January 2023.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, Cell Lines, and Equipment
    • 4. Reagent and Control Preparation
    • 5. Preparation of Study Samples
    • 6. Cell Preparation
    • 7. Experimental Procedure
    • 8. Calculations
    • 9. Acceptance Criteria
    • 10. References
    • 11. Abbreviations
    • 12. Appendix
  • NCL Method ITA-14. Analysis of Nanoparticle Effects on Maturation of Monocyte Derived Dendritic Cells In Vitro: Version 5
    Timothy M. Potter, Edward Cedrone, Barry W. Neun, Susan L. Strobl, Liubov Zaritskaya, and Marina A. Dobrovolskaia.
    Published: November 2022.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, and Equipment
    • 4. Preparation of Reagents
    • 5. Preparation of Test Nanoparticles
    • 6. Isolation of Human PBMC from Leukopack or Whole Blood
    • 7. Rapid Thaw Method of Cryopreserved PBMC
    • 8. Generation of Immature Dendritic Cells
    • 9. Maturation of Dendritic Cells with or without Nanomaterial
    • 10. Analysis of DC Maturation by Flow Cytometry
    • 11. Abbreviations
    • 12. References
    • 13. Appendix
  • NCL Method ITA-40. Understanding the Role of Scavenger Receptor A1 in Nanoparticle Uptake by Murine Macrophages: Version 2
    Edward Cedrone, Martin Schuster, Rosemarie Preyer, and Marina A. Dobrovolskaia.
    Published: October 2022.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, Cell Lines, and Equipment
    • 4. Reagent and Control Preparation
    • 5. Preparation of Study Samples
    • 6. Cell Preparation
    • 7. Experimental Procedure
    • 8. Results and Analysis
    • 9. References
    • 10. Abbreviations
    • 11. Appendix
  • NCL Method ITA-7. Detection of Nitric Oxide Production by the Macrophage Cell Line RAW264.7: Version 2
    Timothy M. Potter, Edward Cedrone, Barry W. Neun, and Marina A. Dobrovolskaia.
    Published: September 2020.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, Cell Lines, and Equipment
    • 4. Reagent and Control Preparation
    • 5. Preparation of Study Samples
    • 6. Cell Preparation
    • 7. Experimental Procedure
    • 8. Calculations
    • 9. Acceptance Criteria
    • 10. References
    • 11. Abbreviations
    • 12. Appendix
  • NCL Method ITA-27. Multiplex Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of Human Cytokines in Culture Supernatants: Version 1
    Edward Cedrone, Timothy M. Potter, Barry W. Neun, Abby Tyler, and Marina A. Dobrovolskaia.
    Published: September 2020.
    • 1. Introduction
    • 2. Principle
    • 3. Reagents, Materials, and Equipment
    • 4. Preparation of Reagents and Controls
    • 5. Preparation of Inhibition/Enhancement Controls (IECs)
    • 6. Experimental Procedure for 4-plex Interferon Multiplex
    • 7. Experimental Procedure for 14-plex and 15-plex multiplex
    • 8. Acceptance Criteria
    • 9. Example of ELISA Plate Template
    • 10. Example of Product Information Card
    • 11. References
    • 12. Abbreviations
  • NCL Method ITA-36. Detection of Naturally Occurring Antibodies to PEG and PEGylated Liposomes in Plasma of Human Donor Volunteers: Version 2
    Barry W. Neun and Marina A. Dobrovolskaia.
    Published: June 2020.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials and Equipment
    • 4. Buffers and Controls
    • 5. Procedure
    • 6. Appendix
  • NCL Method ITA-25. Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of Human IFNγ in Culture Supernatants: Version 3
    Edward Cedrone, Timothy M. Potter, Barry W. Neun, and Marina A. Dobrovolskaia.
    Published: June 2020.
    • 1. Introduction
    • 2. Principle
    • 3. Reagents, Materials, and Equipment
    • 4. Preparation and Storage of Plasma for Preparation of Assay Diluent
    • 5. Preparation of Reagents and Controls
    • 6. Preparation of Calibration Standard and Quality Controls for ELISA
    • 7. Experimental Procedure
    • 8. Acceptance Criteria
    • 9. Example of ELISA Plate Template
    • 10. Abbreviations
  • NCL Method ITA-24. Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of Human TNF-α in Culture Supernatants: Version 3
    Edward Cedrone, Timothy M. Potter, Barry W. Neun, and Marina A. Dobrovolskaia.
    Published: June 2020.
    • 1. Introduction
    • 2. Principle
    • 3. Reagents, Materials, and Equipment
    • 4. Preparation and Storage of Plasma for Preparation of Assay Diluent
    • 5. Preparation of Reagents and Controls
    • 6. Preparation of Calibration Standard and Quality Controls for ELISA
    • 7. Experimental Procedure
    • 8. Acceptance Criteria
    • 9. Example of ELISA Plate Template
    • 10. Abbreviations
  • NCL Method ITA-23. Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of Human IL-1β in Culture Supernatants: Version 3
    Edward Cedrone, Timothy M. Potter, Barry W. Neun, and Marina A. Dobrovolskaia.
    Published: June 2020.
    • 1. Introduction
    • 2. Principle
    • 3. Reagents, Materials, and Equipment
    • 4. Preparation and Storage of Plasma for Preparation of Assay Diluent
    • 5. Preparation of Reagents and Controls
    • 6. Preparation of Calibration Standard and Quality Controls for ELISA
    • 7. Experimental Procedure
    • 8. Acceptance Criteria
    • 9. Example of ELISA Plate Template
    • 10. Abbreviations
  • NCL Method ITA-22. Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of Human IL-8 in Culture Supernatants: Version 3
    Edward Cedrone, Timothy M. Potter, Barry W. Neun, and Marina A. Dobrovolskaia.
    Published: June 2020.
    • 1. Introduction
    • 2. Principle
    • 3. Reagents, Materials, and Equipment
    • 4. Preparation and Storage of Plasma for Preparation of Assay Diluent
    • 5. Preparation of Reagents and Controls
    • 6. Preparation of Calibration Standard and Quality Controls for ELISA
    • 7. Experimental Procedure
    • 8. Acceptance Criteria
    • 9. Example of ELISA Plate Template
    • 10. Abbreviations
  • NCL Method ITA-17. Analysis of Nanoparticle Effects on Leukocyte Procoagulant Activity: Version 3
    Timothy M. Potter, Edward Cedrone, Barry W. Neun, and Marina A. Dobrovolskaia.
    Published: June 2020.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, Cell Lines, and Equipment
    • 4. Reagent and Control Preparation
    • 5. Preparation of Study Samples
    • 6. Cell Preparation
    • 7. Experimental Procedure
    • 8. Calculations
    • 9. Acceptance Criteria
    • 10. References
    • 11. Abbreviations
    • 12. Appendix
  • NCL Method ITA-12. In Vitro Analysis of Nanoparticle Effects on Plasma Coagulation Time: Version 3
    Edward Cedrone, Timothy M. Potter, Barry W. Neun, and Marina A. Dobrovolskaia.
    Published: June 2020.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, and Equipment
    • 4. Preparation of Study Samples
    • 5. Preparation of Test, Normal and Abnormal Control Plasmas
    • 6. Experimental Procedure
    • 7. Calculations and Data Interpretation
    • 8. Acceptance Criteria
    • 9. References
    • 10. Abbreviations
    • 11. Appendix
  • NCL Method ITA-11. Measurement of Nanoparticle Effects on Cytotoxic Activity of NK Cells by Label-Free RT-CES System: Version 3
    Timothy M. Potter, Edward Cedrone, Barry W. Neun, and Marina A. Dobrovolskaia.
    Published: June 2020.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, and Equipment
    • 4. Reagent Preparation
    • 5. Preparation of Study Samples
    • 6. Preparation of Effector and Target Cells
    • 7. Experimental Procedure (This will require 3 days.)
    • 8. Calculations
    • 9. Acceptance Criteria
    • 10. References
    • 11. Abbreviations
  • NCL Method ITA-9.2. In Vitro Assay for Assessing Nanoparticle Effects on Monocyte/Macrophage Phagocytic Function: Version 1
    Timothy M. Potter, Edward Cedrone, Barry W. Neun, and Marina A. Dobrovolskaia.
    Published: May 2020.
    • 1. Introduction
    • 2. Principles and Limitation
    • 3. Reagents, Materials, Cell Lines, and Equipment
    • 4. Reagent and Control Preparation
    • 5. Preparation of Study Samples
    • 6. Cell Preparation
    • 7. Experimental Procedure, Day 1
    • 8. Experimental Procedure, Day 2
    • 9. Calculations
    • 10. Acceptance Criteria
    • 11. References
    • 12. Abbreviations
    • 13. Appendix
  • NCL Method ITA-9.1. Phagocytosis Assay: Version 1.2
    Timothy M. Potter, Edward Cedron, Barry W. Neun, and Marina A. Dobrovolskaia.
    Published: May 2020.
    • 1. Introduction
    • 2. Principles and Limitation
    • 3. Reagents, Materials, Cell Lines, and Equipment
    • 4. Reagent and Control Preparation
    • 5. Preparation of Study Samples
    • 6. Cell Preparation
    • 7. Experimental Procedure
    • 8. Calculations
    • 9. Acceptance Criteria
    • 10. References
    • 11. Abbreviations
    • 12. Appendix
  • NCL Method ITA-8.2. Analysis of Nanoparticle Chemoattractant Properties Using Label-Free, Real-Time Chemotaxis Assay: Version 1.0
    Edward Cedrone, Julia Bui, Naichen Yu, and Marina A. Dobrovolskaia.
    Published: May 2020.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, Cell Lines, and Equipment
    • 4. Reagent and Control Preparation
    • 5. Preparation of Study Samples
    • 6. RAW 264.7 Cell Preparation
    • 7. Fibronectin Coating
    • 8. Experimental Procedure
    • 9. Calculations
    • 10. Acceptance Criteria
    • 11. References
    • 12. Abbreviations
    • 13. Appendix
  • NCL Method ITA-8.1. Chemotaxis Assay: Version 2
    Timothy M. Potter, Edward Cedrone, Barry W. Neun, and Marina A. Dobrovolskaia.
    Published: May 2020.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, Cell Lines, and Equipment
    • 4. Reagent and Control Preparation
    • 5. Preparation of Study Samples
    • 6. HL-60 Cell Preparation
    • 7. Experimental Procedure
    • 8. Calculations
    • 9. Acceptance Criteria
    • 10. References
    • 11. Abbreviations
    • 12. Appendix
  • NCL Method ITA-6.3. Leukocyte Proliferation Assay: Version 2
    Timothy M. Potter, Edward Cedrone, Sarah L. Skoczen, Barry W. Neun, and Marina A. Dobrovolskaia.
    Published: May 2020.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, and Equipment
    • 4. Reagent and Control Preparation
    • 5. Preparation of Study Samples
    • 6. Isolation of Human Lymphocytes
    • 7. Experimental Procedure
    • 8. Calculations
    • 9. Acceptance Criteria
    • 10. References
    • 11. Abbreviations
    • 12. Appendix
  • NCL Method ITA-6.2. Leukocyte Proliferation Assay: Version 2
    Timothy M. Potter, Edward Cedrone, Sarah L. Skoczen, Barry W. Neun, and Marina A. Dobrovolskaia.
    Published: May 2020.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, and Equipment
    • 4. Reagent and Control Preparation
    • 5. Preparation of Study Samples
    • 6. Isolation of Human Lymphocytes
    • 7. Experimental Procedure
    • 8. Calculations
    • 9. Acceptance Criteria
    • 10. References
    • 11. Abbreviations
    • 12. Appendix
  • NCL Method ITA-5.2. Analysis of Complement Activation by Single-Plex EIA or Multiplex ELISA: Version 3
    Barry W. Neun, Edward Cedrone, and Marina A. Dobrovolskaia.
    Published: May 2020.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, and Equipment
    • 4. Reagent and Control Preparation
    • 5. Preparation of Study Samples
    • 6. Plasma Collection and Storage
    • 7. Experimental Procedure for Sample Preparation
    • 8. Experimental Procedure for Single-Plex EIA
    • 9. Experimental Procedure for Multi-plex ELISA
    • 10. Data Analysis
    • 11. Acceptance Criteria
    • 12. References
    • 13. Abbreviations
  • NCL Method ITA-3. Mouse Granulocyte-Macrophage Colony-Forming Unit Assay: Version 4
    Barry W. Neun, Edward Cedrone, and Marina A. Dobrovolskaia.
    Published: May 2020.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, and Equipment
    • 4. Animals
    • 5. Reagent and Control Preparation
    • 6. Preparation of Study Samples
    • 7. Isolation and Counting of Bone Marrow Cells
    • 8. Experimental Procedure
    • 9. Description of CFU-GM
    • 10. Calculations
    • 11. Acceptance Criteria
    • 12. References
    • 13. Abbreviations
  • NCL Method ITA-2.2. Analysis of Platelet Aggregation by Light Transmission Aggregometry: Version 3
    Jamie Rodriguez, Barry W. Neun, Edward Cedrone, and Marina A. Dobrovolskaia.
    Published: May 2020.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, and Equipment
    • 4. Preparation of Plasma, Test Samples and Controls
    • 5. Experimental Procedure
    • 6. Data Analysis and Calculations
    • 7. Acceptance Criteria
    • 8. References
    • 9. Abbreviations
  • NCL Method ITA-2.1. Analysis of Platelet Aggregation by Cell Counting: Version 4
    Barry W. Neun, Edward Cedrone, and Marina A. Dobrovolskaia.
    Published: May 2020.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, and Equipment
    • 4. Preparation of Plasma, Test Samples and Controls
    • 5. Experimental Procedure
    • 6. Calculations and Data Interpretation
    • 7. Acceptance Criteria
    • 8. References
    • 9. Abbreviations
  • NCL Method ITA-1. Analysis of Hemolytic Properties of Nanoparticles: Version 4
    Barry W. Neun, Edward Cedrone, and Marina A. Dobrovolskaia.
    Published: May 2020.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, and Equipment
    • 4. Preparation of Standards and Controls
    • 5. Preparation of Blood and Study Samples
    • 6. Experimental Procedure
    • 7. Calculations and Result Interpretation
    • 8. Acceptance Criteria
    • 9. Example of Nanoparticle Interference
    • 10. References
    • 11. Abbreviations
    • 12. Appendix
  • NCL Method IEA-1. Analysis of Nanoparticle Effects on Invasion of Cancer Cells: Version 1.0
    Julia Bui, Edward Cedrone, Naichen Yu, and Marina A. Dobrovolskaia.
    Published: May 2020.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, Cell Lines, and Equipment
    • 4. Reagent and Control Preparation
    • 5. Preparation of Study Samples
    • 6. MDA-MB-231 Cell Preparation
    • 7. Matrigel Coating of the Upper Chamber of CIM-Plate 16
    • 8. Experimental Procedure
    • 9. Calculations
    • 10. References
    • 11. Appendix
    • 12. Abbreviations
  • NCL Method ITA-35. Antigen-Specific Stimulation of CD8+ T-cells by Murine Bone Marrow-Derived Dendritic Cells: Version 1
    Enping Hong and Marina A. Dobrovolskaia.
    Published: February 2019.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, and Equipment
    • 4. Animals
    • 5. Reagent and Control Preparation
    • 6. Preparation of Nanoparticles
    • 7. Isolation and Counting of Bone Marrow Cells
    • 8. Generation of Immature Dendritic Cells
    • 9. Treatment with Nanoparticles and Controls
    • 10. Extraction and Homogenization of OT-I Spleen
    • 11. CD8+ T-Cell Labeling and Purification
    • 12. Cell Stimulation Experiment
    • 13. Assessing T-Cell Purity
    • 14. Ending Experiment
    • 15. Flow Cytometry
    • 16. Data Analysis and Report
    • 17. References
    • 18. Abbreviations
    • 19. Appendix
  • NCL Method ITA-34. Detection of Antigen Presentation by Murine Bone Marrow-Derived Dendritic Cells: Version 1
    Enping Hong and Marina A. Dobrovolskaia.
    Published: February 2019.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, and Equipment
    • 4. Animals
    • 5. Reagent and Control Preparation
    • 6. Preparation of Nanoparticles
    • 7. Isolation and Counting of Bone Marrow Cells
    • 8. Generation of Immature Dendritic Cells
    • 9. Treatment with Nanoparticles and Controls
    • 10. Flow Cytometry
    • 11. Data Analysis and Report
    • 12. References
    • 13. Abbreviations
    • 14. Appendix
  • NCL Method ITA-33. Detection of Changes in Mitochondrial Membrane Potential in T-cells Using JC-1 Dye: Version 1
    Ankit Shah and Marina A. Dobrovolskaia.
    Published: February 2019.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, and Equipment
    • 4. Reagent and Control Preparation
    • 5. Preparation of Nanoparticles
    • 6. Collection and Handling of Whole Blood
    • 7. Preparation of T-Cells
    • 8. Treatment with Nanoparticles and Controls
    • 9. Flow Cytometry
    • 10. Data Analysis and Report
    • 11. Acceptance criteria
    • 12. References
    • 13. Abbreviations
    • 14. Appendix
  • NCL Method ITA-32. Detection of Mitochondrial Oxidative Stress in T-cells Using MitoSOX Red Dye: Version 1
    Ankit Shah and Marina A. Dobrovolskaia.
    Published: February 2019.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, and Equipment
    • 4. Reagent and Control Preparation
    • 5. Preparation of Nanoparticles
    • 6. Collection and Handling of Whole Blood
    • 7. Preparation of T-Cells
    • 8. Treatment with Nanoparticles and Controls
    • 9. Flow Cytometry
    • 10. Data Analysis and Report
    • 11. Acceptance Criteria
    • 12. References
    • 13. Abbreviations
    • 14. Appendix
  • NCL Method ITA-31. Detection of Nanoparticle-Mediated Total Oxidative Stress in T-Cells Using CM-H2DC-FDA Dye: Version 1
    Ankit Shah and Marina A. Dobrovolskaia.
    Published: February 2019.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, and Equipment
    • 4. Reagent and Control Preparation
    • 5. Preparation of Nanoparticles
    • 6. Collection and Handling of Whole Blood
    • 7. Preparation of T-Cells
    • 8. Treatment with Nanoparticles and Controls
    • 9. Flow Cytometry
    • 10. Data Analysis and Report
    • 11. Acceptance Criteria
    • 12. References
    • 13. Abbreviations
    • 14. Appendix
  • NCL Method ITA-26. Detection of Intracellular Complement Activation in Human T Lymphocytes: Version 1
    Anna N. Ilinskaya, Ankit Shah, and Marina A. Dobrovolskaia.
    Published: February 2019.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, and Equipment
    • 4. Reagent and Control Preparation
    • 5. Isolation of Human PBMC
    • 6. Treatment with Nanoparticles and Controls
    • 7. Preparation of Cells for Flow Cytometry
    • 8. Flow Cytometry
    • 9. Data Analysis and Report
    • 10. Acceptance Criteria
    • 11. References
• Pharmacology/Toxicology protocols
  • Guide to NCL In Vivo Studies: Efficacy, Pharmacokinetics & Toxicology
    Published: September 2022.
    • Introduction
    • Efficacy Studies
    • Toxicity Studies
    • Pharmacokinetic Studies
    • References
  • NCL Method GTA-1. LLC-PK1 Kidney Cytotoxicity Assay: Version 1.3
    Stephan T. Stern, Pavan P. Adiseshaiah, and Timothy M. Potter.
    Published: April 2020.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, and Equipment
    • 4. Reagent and Control Preparation
    • 5. Experimental Procedure
    • 6. Calculations
    • 7. Acceptance Criteria
    • 8. References
    • 9. Abbreviations
    • 10. Appendix
  • NCL Method PHA-1. Radioactive Blood Partitioning Assay: Version 1.1
    Stephan T. Stern.
    Published: June 2017.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, Cell Lines, and Equipment
    • 4. Experimental Procedure
    • 5. Calculations
    • 6. References
    • 7. Abbreviations
  • NCL Method PHA-2. Ultrafiltration Drug Release Assay Utilizing a Stable Isotope Tracer: Version 1.0
    Stephan T. Stern and Sarah Skoczen.
    Published: April 2017.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, and Equipment
    • 4. Experimental Procedure
    • 5. References
    • 6. Abbreviations
  • NCL Method GTA-3. Hep G2 Hepatocyte Glutathione Assay: Version 1.2
    Stephan T. Stern, Timothy M. Potter, and Barry W Neun.
    Published: April 2016.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, Cell Lines, and Equipment
    • 4. Reagent and Control Preparation
    • 5. Experimental Procedure
    • 6. Protein Determination (Bradford Assay)
    • 7. Calculations
    • 8. Acceptance Criteria
    • 9. References
    • 10. Abbreviations
    • 11. Appendices
  • NCL Method GTA-14. Hep G2 Hepatocarcinoma Homogeneous Apoptosis Assay: Version 1.2
    Stephan T. Stern and Barry W. Neun.
    Published: April 2011.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, Cell Lines, and Equipment
    • 4. Reagent and Control Preparation
    • 5. Experimental Procedure
    • 6. Calculations
    • 7. Acceptance Criteria
    • 8. References
    • 9. Abbreviations
    • 10. Appendix
  • NCL Method GTA-12. Autophagic Dysfunction in LLC-PK1 Cells: Version 1.1
    Stephan T. Stern and Barry W. Neun.
    Published: February 2011.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, Cell Lines, and Equipment
    • 4. Reagent and Control Preparation
    • 5. Test sample preparation
    • 6. Experimental Procedure
    • 7. Calculations
    • 8. Acceptance Criteria
    • 9. References
    • 10. Abbreviations
    • 11. Appendix
  • NCL Method GTA-7. Hepatocyte Primary ROS Assay: Version 1.1
    Stephan T. Stern and Banu S. Zolnik.
    Published: April 2010.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, Cell Lines, and Equipment
    • 4. Reagent and Control Preparation (Prepare immediately prior to use)
    • 5. Experimental Procedure
    • 6. Test Nanomaterial Addition
    • 7. Calculations
    • 8. Acceptance Criteria
    • 9. References
    • 10. Abbreviations
    • 11. Appendix
  • NCL Method GTA-6. HepG2 Hepatocarcinoma Apoptosis Assay: Version 1.1
    Stephan T. Stern and Timothy M. Potter.
    Published: April 2010.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, Cell Lines, and Equipment
    • 4. Reagent and Control Preparation
    • 5. Experimental Procedure
    • 6. Calculations
    • 7. Acceptance Criteria
    • 8. References
    • 9. Abbreviations
    • 10. Appendices
  • NCL Method GTA-5. LLC-PK1 Kidney Cell Apoptosis Assay: Version 1.1
    Stephan T. Stern and Timothy M. Potter.
    Published: April 2010.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, Cell Lines, and Equipment
    • 4. Reagent and Control Preparation
    • 5. Experimental Procedure
    • 6. Calculations
    • 7. Acceptance Criteria
    • 8. References
    • 9. Abbreviations
    • 10. Appendices
  • NCL Method GTA-4. Hep G2 Hepatocyte Lipid Peroxidation Assay: Version 1.1
    Stephan T. Stern, Timothy M. Potter, and Barry W Neun.
    Published: April 2010.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, Cell Lines, and Equipment
    • 4. Reagent and Control Preparation
    • 5. Experimental Procedure
    • 6. Protein Determination (Bradford Assay)
    • 7. Calculations
    • 8. Acceptance Criteria
    • 9. References
    • 10. Abbreviations
    • 11. Appendices
• Physicochemical characterization protocols
  • NCL Method PCC-23. Quantitation of Residual DMSO in Nanoformulations Using Gas Chromatography with Direct Injection and Flame Ionization Detection: Version 1
    Krishna Kattel and Jeffrey D. Clogston.
    Published: August 2023.
    • 1. Introduction
    • 2. Reagents and Equipment
    • 3. Instrumentation
    • 4. Evaluation of Method
    • 5. Standard Preparation
    • 6. Sample Preparation
    • 7. Calculations
    • 8. Example, Quantitative Analysis of Residual DMSO in Nanoformulation
    • 9. References
  • NCL Method PCC-22. Residual Organic Solvent Analysis in Nanoformulations Using Headspace Gas Chromatography: Version 1
    Krishna Kattel and Jeffrey D. Clogston.
    Published: September 2022.
    • 1. Introduction
    • 2. Reagents and Equipment
    • 3. Instrumentation
    • 4. Evaluation of Method Sensitivity and Linearity
    • 5. Standard Preparation
    • 6. Sample Preparation
    • 7. Calculations
    • 8. Example, Quantitative Analysis of Residual Ethanol in Doxil (Lot# JHZUA01)
    • 9. References
  • Parameters, Methods and Considerations for the Physicochemical Characterization of Colloidal Metal Nanoparticles
    Published: June 2022.
    • Size/Size Distribution
    • Composition
    • Surface Characteristics
    • Purity
    • Batch-to-Batch Consistency
    • Stability
    • Starting Material Characterization
    • Morphology
    • Relevant NCL Publications
  • Parameters, Methods and Considerations for the Physicochemical Characterization of Polymeric Nanoparticles
    Published: June 2022.
    • Size/Size Distribution
    • Composition
    • Surface Characteristics
    • Purity
    • Batch-to-Batch Consistency
    • Stability
    • Starting Material Characterization
    • Morphology
    • Relevant NCL Publications
  • Parameters, Methods and Considerations for the Physicochemical Characterization of Liposomal Products
    Jeffrey D. Clogston.
    Published: June 2022.
    • Size/Size Distribution
    • Composition
    • Surface Characteristics
    • Purity
    • Morphology
    • Stability
    • Starting Material Characterization
    • Batch-to-Batch Consistency
    • Relevant NCL Publications
  • NCL Method PCC-21. Measuring Size and Number Concentration of Metallic Nanoparticles using SP-ICP-MS: Version 1
    Matthew Hansen and Jeffrey D. Clogston.
    Published: January 2021.
    • 1. Introduction
    • 2. Reagents, Materials and Equipment
    • 3. Methods
    • 4. Data Analysis
    • 5. Precautions and Guidelines
    • 6. Waste Disposal
    • 7. References
    • 8. Abbreviations
  • NCL Method PCC-19. Asymmetric-Flow Field-Flow Fractionation: Version 1.0
    Jeffrey D. Clogston and Yingwen Hu.
    Published: May 2020.
    • 1. Introduction
    • 2. Principle
    • 3. Equipment and Reagents
    • 4. Experimental Procedure
    • 5. Method Application/Examples
    • 6. References
  • NCL Method PCC-18. Quantitation of APIs in Polymeric Prodrug Formulations: Version 1.0
    Jeffrey D. Clogston and Yingwen Hu.
    Published: May 2020.
    • 1. Introduction
    • 2. Reagents and Equipment
    • 3. Measurement Principle
    • 4. Experimental Procedure
    • 5. Data Analysis
    • 6. Safety
    • 7. References
  • NCL Method PCC-2. Measuring Zeta Potential of Nanoparticles: Version 1.2
    Jeffrey D. Clogston and Alison Vermilya.
    Published: April 2020.
    • 1. Introduction
    • 2. Reagents, Materials, and Equipment
    • 3. Methods
    • 4. Precautions and Guidelines
    • 5. Waste Disposal Information
    • 6. References
    • 7. Abbreviations
  • NCL Method PCC-16. Quantitation of PEG on PEGylated Gold Nanoparticles Using Reversed Phase High Performance Liquid Chromatography and Charged Aerosol Detection: Version 1.0
    Jeffrey D. Clogston and Mackensie C. Smith.
    Published: October 2017.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, and Equipment
    • 4. Experimental Procedure
    • 5. Data Analysis
    • 6. References
    • 7. Abbreviations
  • NIST - NCL Joint Assay Protocol, PCC-15. Measuring the Size of Colloidal Gold Nano-particles using High-Resolution Scanning Electron Microscopy: Version 1.0
    András E. Vladár and Bin Ming.
    Published: July 2011.
    • 1. Introduction
    • 2. Sample Preparation
    • 3. Measurement Procedure
    • 4. Image Processing and Analysis
    • 5. Observations of SEM images
    • 6. Statistical analysis of particle size measurement results
    • 7. References
    • 8. Abbreviations
  • NIST - NCL Joint Assay Protocol, PCC-14. Quantification of Free and Chelated Gadolinium Species in Nanoemulsion-Based Magnetic Resonance Imaging Contrast Agent Formulations using Hyphenated Chromatography Methods: Version 1.0
    Danielle Cleveland.
    Published: June 2010.
    • 1. Introduction
    • 2. Reagents and Equipment
    • 3. Reagent Preparation
    • 4. Measurement of Total Gadolinium by ID-ICP-MS
    • 5. Reversed-Phase Liquid Chromatography with Fluorescence Detection and on-line ID-ICP-MS
    • 6. High Pressure Size Exclusion Chromatography with Online ID-ICP-MS Detection
    • 7. High-Pressure Size Exclusion Chromatography SID-ICP-MS Analysis
    • 8. References
    • 9. Abbreviations
    • Appendix A. Uncertainty for ICP-MS Measurements
    • Appendix B. Procedure for Day-to-Day Use of Analytical Balances
  • NIST - NCL Joint Assay Protocol, PCC-13. Measuring the pH of Nanoparticle Suspensions: Version 1.1
    Kenneth W. Pratt.
    Published: March 2010.
    • 1. Introduction
    • 2. Reagents and Equipment
    • 3. Experimental Procedure
    • 4. Calculations
    • 5. Acceptance Criteria
    • 6. References
    • 7. Abbreviations
  • NIST - NCL Joint Assay Protocol, PCC-12. Measuring the Electrolytic Conductivity of Nanoparticle Suspensions: Version 1.1
    Kenneth W. Pratt.
    Published: March 2010.
    • 1. Introduction
    • 2. Reagents and Equipment
    • 3. Experimental Procedure
    • 4. Calculations
    • 5. Acceptance Criteria
    • 6. References
    • 7. Abbreviations
  • NIST - NCL Joint Assay Protocol, PCC-10. Analysis of Gold Nanoparticles by Electrospray Differential Mobility Analysis (ES-DMA): Version 1.1
    Leonard F. Pease, III, De-Hao Tsai, Rebecca A. Zangmeister, Michael R. Zachariah, and Michael J. Tarlov.
    Published: March 2010.
    • 1. Introduction
    • 2. Reagents and Equipment
    • 3. Reagent Preparation
    • 4. Preparation of Samples
    • 5. Analysis of Sample by ES-DMA used the following conditions
    • 6. Data Analysis
    • 7. References
    • 8. Abbreviations
  • NIST - NCL Joint Assay Protocol, PCC-9. Determination of Gold in Rat Blood with Inductively Coupled Plasma Mass Spectrometry: Version 1.1
    L.L. Yu, L.J. Wood, and S.E. Long.
    Published: February 2010.
    • 1. Introduction
    • 2. Reagents and Equipment
    • 3. Digestion of Blood
    • 4. Preparation of Analytical Portions
    • 5. ICP-MS Measurements
    • 6. Calculations
    • 7. Abbreviations
    • Appendix A. Setup and Optimization Procedure for Agilent 7500cs ICP Mass Spectrometer
    • Appendix B. Procedure for Use of Balances
    • Appendix C. Microwave Digestion
  • NIST - NCL Joint Assay Protocol, PCC-8. Determination of Gold in Rat Tissue with Inductively Coupled Plasma Mass Spectrometry: Version 1.1
    L.L. Yu, L.J. Wood, and S.E. Long.
    Published: February 2010.
    • 1. Introduction
    • 2. Reagents and Equipment
    • 3. Digestion of Tissue
    • 4. Preparation of Analytical Portions
    • 5. ICP-MS Measurements
    • 6. Calculations
    • 7. Abbreviations
    • Appendix A. Setup and Optimization Procedure for Agilent 7500cs ICP Mass Spectrometer
    • Appendix B. Procedure for Use of Balances
    • Appendix C. Microwave Digestion
  • NIST - NCL Joint Assay Protocol, PCC-7. Measuring the Size of Nanoparticles Using Transmission Electron Microscopy (TEM): Version 1.1
    John E. Bonevich and Wolfgang K. Haller.
    Published: February 2010.
    • 1. Introduction
    • 2. Sample Preparation Procedures
    • 3. Measurement Procedures
    • 4. Precautions and Guidelines
    • 5. Performance Verification and Data Analysis
    • 6. Reporting Particle Size Data
    • 7. References
    • 8. Glossary of Terms
    • 9. Abbreviations
  • NIST - NCL Joint Assay Protocol, PCC-1. Measuring the Size of Nanoparticles in Aqueous Media Using Batch-Mode Dynamic Light Scattering: Version 1.1
    Vincent A. Hackley and Jeffrey D. Clogston.
    Published: February 2010.
    • 1. Introduction
    • 2. Sample Preparation and Measurement Procedure
    • 3. Precautions and Guidelines
    • 4. Performance Verification and Data Analysis
    • 5. Reporting Particle Size Data
    • 6. References
    • 7. Glossary of Terms
    • 8. Abbreviations
  • NIST - NCL Joint Assay Protocol, PCC-6. Size Measurement of Nanoparticles Using Atomic Force Microscopy: Version 1.1
    Jaroslaw Grobelny, Frank W. DelRio, Namboodiri Pradeep, Doo-In Kim, Vincent A. Hackley, and Robert F. Cook.
    Published: October 2009.
    • 1. Introduction
    • 2. Sample Preparation and Inspection
    • 3. AFM Imaging and Size Measurement Procedure
    • 4. Image Analysis
    • 5. Reporting Particle Size Data
    • 6. References
    • 7. Abbreviations
• Sterility and endotoxin protocols
  • Endotoxin & Depyrogenation Tips
    Published: June 2023.
    • Endotoxin and Depyrogenation Tips
    • References
  • NCL Method STE-3. Detection of Mycoplasma Contamination in Nanoparticle Formulations: Version 3
    Timothy M. Potter, Barry W. Neun, and Marina A. Dobrovolskaia.
    Published: September 2020.
    • 1. Introduction
    • 2. Principle
    • 3. Preparation of study samples
    • 4. Reagents and Equipment
    • 5. Preparation of cells for mycoplasma testing
  • NCL Method STE-2.4. Detection of Bacterial Contamination using Tryptic Soy Agar Plates and RPMI Suspension Tests for the Purposes of Determining Bacterial ID: Version 2
    Barry W. Neun, Edward Cedrone, Timothy M. Potter, and Marina A. Dobrovolskaia.
    Published: September 2020.
    • 1. Introduction
    • 2. Reagents, Materials, and Equipment
    • 3. Reagent Preparation
    • 4. Preparation of Controls
    • 5. Study Samples
    • 6. Experimental Procedure
    • 7. Interpretation of Results and Acceptance Criteria
    • 8. Abbreviations
  • NCL Method STE-2.3. Detection of Bacterial Contamination Using Tryptic Soy Broth Agar Plates: Version 3
    Barry W. Neun, Timothy M. Potter, and Marina A. Dobrovolskaia.
    Published: September 2020.
    • 1. Introduction
    • 2. Reagents, Materials, and Equipment
    • 3. Reagent Preparation
    • 4. Preparation of Controls
    • 5. Study Samples
    • 6. Experimental Procedure
    • 7. Interpretation of Results and Acceptance Criteria
    • 8. Abbreviations
  • NCL Method STE-2.2. Detection of Bacterial Contamination Using Luria Broth Agar Plates: Version 3
    Barry W. Neun, Timothy M. Potter, and Marina A. Dobrovolskaia.
    Published: September 2020.
    • 1. Introduction
    • 2. Reagents, Materials, and Equipment
    • 3. Reagent Preparation
    • 4. Preparation of Controls
    • 5. Study Samples
    • 6. Experimental Procedure
    • 7. Interpretation of Results and Acceptance Criteria
    • 8. Abbreviations
  • NCL Method STE-2.1. Detection of Microbial Contamination Using Millipore Sampler Devices: Version 3
    Barry W. Neun, Timothy M. Potter, and Marina A. Dobrovolskaia.
    Published: September 2020.
    • 1. Introduction
    • 2. Reagents, Materials, and Equipment
    • 3. Reagent Preparation
    • 4. Preparation of Controls
    • 5. Study Samples
    • 6. Experimental Procedure
    • 7. Interpretation of Results and Acceptance Criteria
    • 8. References
    • 9. Abbreviations
  • NCL Method STE-4. Detection and Quantification of β-(1,3)-D-Glucan Contamination in Nanoparticle Formulations by Factor C Depleted LAL (Glucatell®) Assay: Version 1
    Barry W. Neun and Marina A. Dobrovolskaia.
    Published: June 2020.
    • 1. Introduction
    • 2. Principles
    • 3. Reagents, Materials, and Equipment
    • 4. Reagent Preparation
    • 5. Preparation of Standard Curve and Quality Controls
    • 6. Preparation of Study Samples
    • 7. Experimental Procedure
    • 8. Assay Acceptance Criteria
    • 9. Data Interpretation
    • 10. References
    • 11. Abbreviations
    • 12. Appendix