Assay Cascade Protocols
Frederick National Lab
Nanotechnology Characterization Laboratory

Physicochemical Characterization					Method ID
	Size, Size Distribution
		Measuring the Size of Nanoparticles in Aqueous Media Using Batch-Mode DLS See the DLS Video Protocol demonstration			NIST-NCL PCC-1
		Size Measurement of Nanoparticles Using Atomic Force Microscopy			NIST-NCL PCC-6
		Measuring the Size of Nanoparticles Using Transmission Electron Microscopy			NIST-NCL PCC-7
		Analysis of Gold Nanoparticles by Electrospray Differential Mobility Analysis			NIST-NCL PCC-10
		Measuring the Size of Colloidal Gold Nanoparticles Using High-Resolution Scanning Electron Microscopy			NIST-NCL PCC-15
	Topology Molecular Weight Aggregation Purity Chemical Composition
		Determination of Gold in Rat Tissue with Inductively Coupled Plasma-Mass Spectrometry			NIST-NCL PCC-8
		Determination of Gold in Rat Blood with Inductively Coupled Plasma-Mass Spectrometry			NIST-NCL PCC-9
		Method for Determination of the Mass Fraction of Particle-Bound Gold in Suspensions of Gold Nanoparticles			NIST-NCL PCC-11
		Quantification of Free and Chelated Gadolinium Species in Nanoemulsion-Based Magnetic Resonance Imaging Contrast Agent Formulations using Hyphenated Chromatography Methods			NIST-NCL PCC-14
	Surface Characteristics Functionality Zeta Potential
		Measuring Zeta Potential of Nanoparticles			PCC-2
		Measuring the Electrolytic Conductivity of Nanoparticle Suspensions			NIST-NCL PCC-12
		Measuring the pH of Nanoparticle Suspensions			NIST-NCL PCC-13
	Stability Solubility
In Vitro Characterization					Method ID
	Sterility
		Detection of Endotoxin Contamination
			End Point Chromogenic LAL Assay		STE-1.1
			Kinetic Turbidity LAL Assay		STE-1.2
			Gel-Clot LAL Assay		STE-1.3
		Detection of Microbial Contamination			STE-2
		Detection of Mycoplasma Contamination			STE-3
	Targeting
		Cell Binding/Internalization
	In Vitro Immunology
		Blood Contact Properties
			Analysis of Hemolytic Properties of Nanoparticles		ITA-1
			Analysis of Platelet Aggregation		ITA-2
			Analysis of Nanoparticle Interaction with Plasma Proteins by 2D PAGE		ITA-4
			Qualitative Analysis of Total Complement Activation by Western Blot		ITA-5.1
			Quantitative Analysis of Complement Activation		ITA-5.2
			Coagulation Assay		ITA-12
		Cell-Based Assays
			Mouse Granulocyte Macrophage Colony-Forming Unit Assay		ITA-3
			Leukocyte Proliferation Assay		ITA-6
			Macrophage/Neutrophil Function (4 categories):
				Detection of Nitric Oxide Production by RAW 264.7 Macrophage Cell Line	ITA-7
				Chemotaxis Assay	ITA-8
				Phagocytosis Assay	ITA-9
			Measurement of Nanoparticle Effects on Cytotoxic Activity of NK Cells by Label-Free RT-CES System		ITA-11
			Analysis of Nanoparticle Effects on Maturation of Monocyte Derived Dendritic Cells In Vitro		ITA-14
			In Vitro Induction of Leukocyte Procoagulant Activity by Nanoparticles		ITA-17
	Toxicity
		Oxidative Stress
			Hep G2 Hepatocyte Glutathione Assay		GTA-3
			Hep G2 Hepatocyte Lipid Peroxidation Assay		GTA-4
			Hepatocyte Primary ROS Assay		GTA-7
		Cytotoxicity (necrosis)
			LLC-PK1 Kidney Cytotoxicity Assay (MTT and LDH Release)		GTA-1
			Hep G2 Hepatocarcinoma Cytotoxicity Assay (MTT and LDH Release)		GTA-2
		Cytotoxicity (apoptosis)
			LLC-PK1 Kidney Apoptosis Assay (Caspase 3 Activation)		GTA-5
			Hep G2 Hepatocarcinoma Apoptosis Assay (Caspase 3 Activation)		GTA-6
			Hep G2 Hepatocarcinoma Homogeneous Apoptosis Assay (Caspase 3/7 Activation)		GTA-14
		Autophagy
			Autophagic Dysfunction Assay: Qualitative Analysis of MAP LC3I to LC3-II Conversion by Western Blot		GTA-11
			Autophagic Dysfunction in LLC-PK1 Cells		GTA-12
In Vivo Characterization
	Efficacy
		Therapeutic
		Imaging
	Disposition study
		Tissue Distribution
		Clearance
		Half-life
		Systemic exposure (plasma AUC)
	Single and Repeat-Dose Toxicity
	Immunotoxicity