Cell Biology: Centrifugation Techniques

Instrumental Analytics

• The course covers instrumental analysis, with a focus on applications
• The instructor is Harald Hundsberger at the University of Applied Sciences, Krems
• The semester is Wintersemester 23/24

INSA Overview

• Techniques like centrifugation, UV/Vis spectroscopy, fluorescence spectroscopy, chromatografy, electrophoresis, and mass spectroscopy are covered
• Applications include kinetic and endpoint measurements, RT-PCR, HTS, sequencing, CE, laser scanning microscopy, and more
• Principles of IEX, FPLC, HPLC, electrophoresis/CE, 2D electrophoresis, and mass spectroscopy are addressed

Flow Cytometry

• The lecture focuses on applications of flow cytometry, despite covering the instruments
• Measurements include forward and side scatter
• Forward scatter measures cell size, and side scatter measures internal complexity
• A relevant online resource is provided: https://oncohemakey.com/principles-of-flow-cytometry/

Part I Centrifugation

• A technique widely used in labs and academia
• Various centrifuge types are used for diverse applications
  • Benchtop centrifuges: low volumes, low speeds
  • Larger centrifuges: higher volumes (liters)
  • Ultracentrifuges: high G-forces, vacuum applied (100,000 rpm) for subcellular component separation
  • Elutriation centrifuges: separation by size

Centrifugation - Types of Centrifuges

• Benchtop centrifuges are used for low-volume, low-speed applications.
• Larger centrifuges are used for higher sample volumes (up to several liters).
• Ultracentrifuges use vacuum and high rotational speeds (100,000 rpm) to separate subcellular components due to high G-forces.
• Elutriation centrifuges separate cells by size.

THE CENTRIFUGE

• Centrifugation separates a homogenate into components
• The technique uses rotors (fixed-angle or swinging-arm) with varying capacities and pellet evenness
• Centrifuge speed and time are crucial for successful separation
• Refrigerated and evacuated chambers are essential for high speeds to avoid heating during centrifugation and for preventing rotor imbalance

Safety Considerations UC

• Using specified rotors and sample holders is crucial for safety
• Sample weight and balance are critical, often requiring sub-milligram accuracy
• Equipment has specified lifetimes

Classification on Rotor Type

• Fixed-angle rotors hold larger quantities than swinging-arm rotors
• With fixed-angle rotors pellets form less evenly

Centrifugation - Basics

• Svedberg equation (V = d² (Pp-Pm)g / 18η) relates sedimentation speed (V) to particle size (d), particle density (Pp), medium density (Pm), medium viscosity (η), and relative centrifugal acceleration (g)
• Particle size is crucial for sedimentation, along with density and viscosity

Density-Gradient Centrifugation

• Gradients (often sucrose) separate particles based on density
• The highest density of the medium needs to be higher than the separated particles

Media for density Gradients

• Cesium chloride (CsCl): high osmolarity, used for nucleic acids separation (advantage of low viscosity, disadvantage of high osmolarity)
• Sucrose: non-ionic, no interactions with biological material (advantage), low resolution (disadvantage)
• Percoll: good osmolarity capabilities for whole organelle preparations

Applications for Centrifugation

• Cell harvesting (e.g., yeast, bacteria from fermentations)
• Protein purification
• Desalting and concentrating proteins (Amicon tubes)
• Nucleic acid pellet (Plasmid prep, RNA prep)
• Spin columns (Silica based)
• Subcellular fractionation
• Cell separation (elutriation)

Part II - Spectroscopy Basics

• Little sample prep needed
• Well-defined laboratory equipment is utilized
• UV/Vis, CD, fluorescence

Optical Spectroscopy

• Requires minimal sample preparation
• Standardization is key
• Applications in biological disciplines include structure determination (CD), concentration measurement (UV/Vis, Fluorescence), bound/unbound state analysis (Fluorescence Polarization), reaction kinetics (Fluorescence, Absorbance), and substance analysis (Absorbance, IR-Spectroscopy).

What is Light?

• Light is an electromagnetic wave with oscillating electric and magnetic fields
• Light exhibits wave-particle duality

Polarized Light

• Light can vibrate in various directions
• Plane-polarized light vibrates in a single direction.
• Filter, vibration direction.
• Circularly polarized light vibrates in a circular pattern
• Elliptical polarization vibrates in an ellipse.

Electro Magnetic Spectrum

• The electromagnetic spectrum spans a broad range of wavelengths at varying frequencies and energies
• UV light can cause sunburn

Interaction of Light and Material/Sample

• Photons interact with material by raising electrons to higher energy levels
• Transitions between energy levels are extremely fast
• Light can be absorbed without subsequent radiation after the initial energy exchange, resulting in excitation
• Excited states exist for finite times and are unstable

Part III - UV/Vis/NIR Spectroscopy

• UV/Vis/NIR spectroscopy deals with measurement of specific absorbance of light at particular wavelengths

Basics of Photometry

• Photometry measures specific absorbance (OD) of light
• Colored liquids absorb specific wavelengths
• Beer-Lambert law relates absorbance to concentration.

Photometric Measurements

• Concentration determination through spectrophotrometric analysis
• Path length measurements require standardized 1cm cuvettes
• Standard curves aid in determining molar extinction coefficients when not known.

Photometric Measurements (continued)

• Absorbance measures light intensity after passing through a sample, using optical density (OD) values
• Optical density (OD): measures absorbance using a logarithmic scale (A = -log T) and can be derived from the transmission of light through the sample.

Relationship Transmission and OD

• Transmission (T) and absorbance (OD) are inversely related
• Different percentages of transmission equate to different OD readings

Classification of Photometric Devices

• Different photometers include single-beam, dual-beam instruments, with varied monochromators and detectors
• Monochromators select wavelengths, and single photodiodes/diode arrays measure transmitted light

Specifications

• Provided technical specifications for DU 800 performance

Applications UV/Vis Spectroscopy

• Protein quantification (Biuret, Bradford, Lowry, BCA methods)
• DNA quantification
• Enzyme kinetics measurement at 340nm, using NADH cofactor
  • Proteases: use available colorimetric substrates

Applications UV/Vis Spectroscopy (continued)

• Cell viability measurements using tetrazolium salts (XTT)
• ELISA (Enzyme-Linked Immunosorbent Assay)

Measurement Modes (Single Wavelength)

• Measures absorbance or optical density at a single wavelength
• Necessary background or blank measurement to account for light scattering

Measurement Modes (Dual Wavelength)

• Measures absorbance (optical density) at two wavelengths simultaneously
• Used for compensation in assays like ELISA
• Employing substrates for alkaline phosphatase (AP) including pNPP (405 nm) and aminoantipyrene/phenyl phosphate (492 nm), with reference wavelengths (620-650nm).

Part V - Circular Dichroism Spectroscopy

• CD determines absorbance differences of left and right circularly polarized light when light is passed through a chiral molecule
• CD analysis typically involves scans from 190-240nm to yield secondary structure information

Circular Dichroism Spectroscopy (continued)

• Chiral molecules exhibit different absorption of left and right circularly polarized light
• CD provides spectral information about the secondary structure of protein structures (helices, sheets, turns, coils)
• CD measurements expressed in ellipticity

Biosimilar Analytics, Rituximab

• UV/Vis spectroscopy used in biosimilar analytics to assess properties of Rituximab (a monoclonal antibody)