L7 Cell Based Assays

What does PAT stand for?

Process Analytical Technology

What does PAT do?

System used for designing, analysing & controlling manufacturing processes through timely measurements of critical quality & performance attributes of raw & in-process materials

What is In-line monitoring?

Real time measurement of process parameters or quality attributes directly in the manufacturing process itself without interrupting flow of production

What is At-line monitoring?

Taking measurements or samples from production process but outside the primary production line

Name some protein characterisation techniques

FACS, Uv-Vis, Circular Dichroism, DLS, SLS, FT-NIR Spectroscopy

Name some common non-specific readout cell assays

Cell number, cell type - morphology, cell viability - live Vs dead, proliferation rate and total biomass

How to measure cell numbers

Counting, normalisation and biomass growth

Methods to measure cell numbers

1. Counting Chambers

2. Cytometers

3. FACS

4. Impedance Spectroscopy

How does impedance spectroscopy work?

- Sample chambers with specially profiled electrodes

- Measure changes in capacitance Vs frequency (voltage sweep)

- Determine cell density (bulk biomass)

What does FACS stand for?

Fluorescence Activated Cell Sorting

What does FACS do?

Sorts cells based on their individual properties such as size, density, complexity and fluorescence intensity

Different techniques to measure cell populations?

- Biochemical assays: western blot, enzymatic, sequencing, metabolic

- Spectroscopy: Abs/Flu/BL/CL

- Cell-on-Chip & biosensor systems

- Mass Spectrometry

Techniques for single cell analyses?

- Cell counting devices

- Automated microscopy

- FACS

- Single cell omics

2 methods for cell morphology detection

1. microscopy - automated or visual

2. flow cytometry - fwd & side scattering

What are the measurement modes?

- Fixed/lysed cells VS Live cells

- End-point VS real time monitoring

- Quantitative VS Qualitative

- One parameter VS multi-parametric

functional parameters & markers of cell based assays?

- Total ATP - non-specific toxicity, viability

- Cell death mechanisms: caspases, Annexin V

- Intracellular markers: Ca2+, pH, MMP (mitochondrial Membrane Potential), ROS

- Signalling events

- Metabolic fluxes

What are the 3 types of cell proliferation & staining assays?

1. 3H-U or BrU incorporation

2. Trypan Blue (abs)

3. DAPI or Hoechst

What is 3H-U assay?

A cell proliferation assay

- Radioactive counting or immunostaining of fixed cells

What is Trypan Blue (abs) assay?

- A membrane integrity probe

- Azo dye from textiles industry, anionic

- Doesn't penetrate cells with intact membranes, stains damaged cells by protein binding

- Visual or automated colorimetric readout

- Crude cell viability assay - dye exclusion

What is DAPI or Hoechst assay?

- Cell permeant fluorescent stains

- Nuclear DNA

- Counter staining

Metabolic Assay?

MTT assay

What does MTT assay measure?

MTT assay measures the activity of mitochondrial dehydrogenases, enzymes present in metabolically active cells

What is the MTT assay based on?

Based on the reduction of MTT, a yellow tetrazolium salt by mitochondrial dehydrogenases in viable cells to form insoluble purple formazan crystals

How many steps in the assay?

2 step assay, replacement to radioactivity assay (3H-U)

What type of incubation?

Live cell incubation & solvent addition

What's diverted in MTT assay?

Diverts cellular NADH to MTT reduction

What is cellular ATP BL Assay?

1 step mix and measure bioluminescent assay

What is required in BL assay?

Cell lysis & recombinant luciferase cocktail

What does luciferase do?

Luciferase catalyses the oxidation of lucerferin in the presence of ATP which produces light

What is used to measure BL assay activity?

Luminometer used to measure light emission, detects & quantifies light signal

What is annexin V apoptosis assay

method to detect & quantify apoptosis in cells

What is annexin V

Annexin V is a 30 kD Ca2+- binding protein, assay relies on ability of annexin V to bind to PS (phosphatidylserine) residues that become exposed on outer leaf of plasma membrane during early stages of apoptosis

What is the principle of annexin v apoptosis

PS translocation to outer membrane leaflet during apoptosis

How are apoptotic cells detected?

Fluorescent labels (FITC) help trace AV & apoptotic cells

What are caspases?

family of cysteine proteases that play central role in apoptosis - cleave specific cellular proteins & dismantle the cell

What are the major & secondary metabolic pathways

Major - Glycolytic, TCA, OXPHOS

Secondary: FA oxidation, PPP, glutaminolysis

What are the bulk markers

In - Oz, glucose

Out - CO2, lactate, H+

How are metabolic fluxes measured?

OXPHOS Flux: OCR - Oxygen Consumption Rate (d[O2]/dt)

Glycolytic Flux: Extracellular acidification (d[H+]/dt)

TCA Activity: CO2 release (d[CO2]/dt, d[H+]/dt)

What are the pharmacological treatments for metabolic fluxes

OXPHOS & ETC: uncoupling, rotenone, antimycin A

Glycolysis: 2-DG, oxamate, galactose

What is the seahorse XF platform

- Specially profile TC microplate

- moving pistons for sealing

- 2 sensor coatings on each piston ( O2 sensor for OCR & pH sensor for ECAR)

- Injection port for compounds

cell normalisation methods in bioassays?

Direct: cell counting

Indirect:

- Total ATP - require lysis, fluctuates

- Total DNA - cell number rather than volume

- Total protein - cell volume

- Total biomass - non specific

What is Z factor

relates positive & negative readings & S.D values

Whats an ideal Z factor

Z =1

Whats the Z factor for cell based assays

Z = 0.5-0.8