Analytical Cytometry Flashcards
What are inline measurements?
Measurements performed in an automated manner inside the bioreactor
What are are offline measurements?
Measurements performed manually. They require sampling
What’s a sensor in terms of bioprocessing?
A device that detects and responds to some type of input from the physical environment. The specific input could be light, heat, motion, moisture, pressure and etc.
What are fluorophores?
A fluorescent chemical compound that can re emit light upon light excitation
What’s flow cytometry?
A technique used to detect and measure physical and chemical characteristics of a population of cells or particles.
What are the 4 phases of the cell growth cycle and where do they happen in terms of high or low cell count vs time?
- Lag phase - low cell count low time
- Exponential (log) phase - increase by a steep upward curve from lag phase
- Stationary phase - highest cell count in cycle at midway on time axis increase from exponential phase. There’s a period of stationary cell count
- Death phase - decrease from stationary phase by approximately the same amount of increase from exponential to stationary . Straight line
Why is it important to track the biomass?
Optimise the culture
- induce systems at best time
- calculate accurate feed rate times
- harvest the product at the correct time
What properties can be measured when looking at the cytology of bacterial cells?
Glucose, ph, do2 -> process control (sensor)
Total cell count
Optical density
Dry cell weight
Viable cell count - CFU,methylene blue
Others:
Metabolomics
Genomics
Proteomics
Transcriptomics
What is optical density used to measure?
Measure only of proliferation
Rarely takes into account the medium composition or changes to medium composition
What does dry cell weight measure and how long till results are available
Measure only of proliferation
Results available at least 12h later
What’s specific gravity measurement for?
Relative measure of how a process is proceeding
What is measured when looking at the cytology for mammalian cells?
Glucose and metabolites (lactate, ammonia)
Ph, do2
Total cell count
Viable cell count - using strains such as trypan blue,DAPI, propidium iodide which penetrate damaged cell membranes
Others:
Metabolomics
Genomics
Proteomics
Transcriptomics
What are omic technologies?
Universal detection of
Proteomics
Metabolomics
Genomics
Transcriptomics - DNA , mRNA, microarrays
Assume homogeneity
Huge amounts of data
Explain manual cell counting
Haemocytometer staining and trypan blue staining
Trypan blue is negatively charged due that only stains cells with compromised cell membrane so indicates cell death
Dead cells are stained blue
Explain automated cell counting
Nucleocounter
- uses either multiple chamber slides or via-1 cassettes
- staining with acridine orange (live cells) and DAPI (damages membranes)
Acridine orange is an organic compound used as nucleic acid-selective fluorescent cationic dye useful for cell cycle determination. Being cell permeable, it interacted with DNA and RNA by intercalation or electrostatic attractions, respectively
What does automated cell counting rely on and assume?
Relies on software estimation
Assumes homogeneity
What are some indirect methods of monitoring cell growth and viability?
Indirect methods
- luminescent atp monitoring
- fluorescent proliferation assays: presto blue, alamar blue
- colorimetric proliferation assays: XTT, MTT
How does calcein work
virtually non fluorescent and once it enters live cells under effect of intracellular enzymes, is converted to polyanionic dye calcein that is well retained within living cells
Producing an intense green fluorescence
Explain what ethidium homodimer does?
Enters cells with damaged membranes and undergoes 40 fold enhancement of fluorescence upon binding to nucleic acids
producing a bright red fluorescence in dead cells
Explain the process of proliferation assays and give a disadvantage
- Grow cells in plate wells
- Add prestoBlue reagent and incubate for 10 mins
- Measure fluorescence (red)
- Measure fluorescence (green)
Requires precise control and is time-consuming
Can use either presto blue or almar blue
Requires calibration c
List the types of assays and their properties
PrestoBlue - resazurin based - single addition - >=10mins - absorbable or fluorescence - live - is compatible with phenol red - 12 cells per well
AlmarBlue - resazurin based - single addition - 1-4 hours - absorbable and fluorescence- live - is compatible - 50 cells/wells
Resazurin powder- resazurin based - weigh powder,prepare solution + single addition -4h - absorbable or fluorescence- live cells - is compatible 100 cells/well
MTT powder - tetrazolium based - weigh powder, prepare solution, + 2 additions, absorbance, cell format is lysis/endpoint, not compatible with phenol red, 1000 cells/well
XTT powder - tertrazolium based, weigh powder, prepare solution, + 2 additions, 2-4 hours, absorbance, lysis/endpoint, compatible, 1000 cells/well
Equipment used - spectrometer, photometer, micro plate reader
What aspects of cell metabolism can be monitored and why?
Glucose
Glutamine
Lactate
Ammonia
Glutamate
Lactate dehydrogenase (LDH)
This monitoring provides insight to creating an a optimised informed feeding regime
How can we measure the cell metabolites?
Using bioanalysers
Cedex HT
Nova biomedical
What is microscopy?
Magnifying cells to view them,
Optical / light microscopy
- bright field
- phase contrast
- DIC
Fluorescence microscopy
Explain a optical/light microscope
Uses photons (light)
Living or fixed sample
3D
Quick
Inexpensive
Up to 1500x magnification
Contrast techniques-> bright field, phase contrast, DIC
options for complex analysis -> time lapse and z stacking
What does DIC stand for?
Differential interference contrast
What is time lapse microscopy used for?
Observing live cells like culture or tissue samples
Growing life forms
Can use a CELL IQ or a bio station (Nikon)
^ these are incubators with built in microscopes can record over set periods of time
Can be used for differentiation
Only works for adherent cells only 2D
What is fluorescence microscopy?
Inverted microscope that uses fluorescence
Surrounded by a temperature control chamber
Fluoresces different colours red, green, blue and yellow
Ideal for live cell imaging and time lapsing
What are fluorophores?
Fluorophores absorb energy (short wavelength) and emit at a longer wavelength (strokes shift)
Filters let us see the individual light from each dye
We can attach fluorophores to molecules/areas of interest
Name 2 fluorophores
Dylight 405
Alexa fluor 405
Pacific blue
FITC
What is immunofluorescemce staining?
Technique to assess expression of specific cell markers (intracellular or surface)
Utilises antibodies and fluorophores
Imaging is done on a fluorescence microscope
What’s direct and indirect labelling when concerned with immunofluorescemce staining?
Direct labelling
- attacking labelling antibodies directly to the cell receptors
- usually used MAb for specificity
Indirect labelling
- attaching labels to antibodies that attach to cell receptors
- 2 steps -> primary and secondary antibodies
- primary - usually MAb for specificity
Secondary - usually PAb for increased sensitivity. Carries the fluoroscope
Name a few benefits of microscopy
Provides morphological data
Can be used for ultrastructure
1) electron microscopy
2) immuno-gold labelling
Give the limitations of microscopy
Assumption of homogeneity (population vs single cell measurement)
Concerns with methods themselves
- viable, but not culturable cells
- false positives - need to understand the technique and any pitfalls
- human input
- time consuming, offline
Ideal analytical methods
- online or in lines
- rapid
- high throughout
- automated
- help you control your process
What’s kind of sensors and probes can be found in a bioreactor?
Standard probes :
- PH probe
- Temperature probe
- DO (dissolved oxygen) probes
What specialised probes are used for cell cultures?
Biomass probes
- measure Optical Density
- measure impedance
What do the Hamilton incyte and dencytee probes measure?
Incyte
- measures permitivity of viable cells
- the signal can be correlated to the viable cell density measured offline
- can be used directly in the bioreactor for real time process control, eliminating the need for offline sampling
Dencytee
- total cell density measurement
- based in optical density or the turbidity of a suspension at NIR wavelength
What does the futura sensor do ?
Measures the impedance of variable cells at radio frequencies
it involves applying an electrical field to polarise cells
The signal can be correlated to the viable cell density or biomass
Can be used directly in the bioreactor for real time process control, eliminating the need for offline sampling
How does measuring the impedance work?
When an electrical field is applied to cells in an aqueous, ionic solution the ions in the solution are forced to move
Ions both inside and outside the cells move until they encounter the plasma membranes, which will act as insulating physical barriers to prevent further movement
Bulk movement causes a charge separation or polarisation of the cells
The magnitude of resulting field induced separations is measured by capacitance
As the volume fraction of cells in a suspension increases, the number of polarised membranes also increases to yield a higher measured capacitance
Dead cells and non biomass solids have no intact plasma membranes and so do not polarise significantly, nor do they contribute significantly to the capacitance of a cell suspension
What’s flow cytometry?
A technique used to detect and measure physical and chemical characteristics of a population of cells or particles. A sample containing cells or particles is suspended in a fluid injected into the flow cytometer instrument
Allows large populations to be studied
Small changes can be significant
More quantitative than microscopy
Quicker and more consistent
What are the advantages of flow cytometry?
Highly versatile
Highly sensitive
Real time, statistically reliable information
Multiple parameter can be measured
Cell number
Cell structure
Protein expression
Cellular metabolic activity -> cell physiology and viability
Individual cell level
Allows informed decision about a process to be made
What are the various variations of flow cytometry specifications?
Can be single sample or high throughput
Multiple lasers can be 3,4,5
Detection of up to 16 colours
Particle size microns to nano meters
With it without cell sorting
What is forward and side scatter?
Cells or particle passing through the beam scatter light which is detected as forward scatter and side scatter
Forward scatter correlates with cell size and as is proportional to the granularity of the cells
Cell population can often distinguish based on differences in their size and granularity alone
Considering blood sample as an example explain how Yh front scatter and back scatter would behave
Larger and more granular granulocyte cells produce a large population with high side scatter and front scatter
Monocytes are large cells but not so granular so these produce a separate population with higher front scatter and lower side scatter
Smaller lymphocytes and lymphoblasts produce a separate population with less front scatter. They are not granular cells so have low side scatter
What are the two types of fluorophores?
FITC (ex/Em) : 495/519nm - green
PE (ex/Em) : 496/578 nm - yellow/orange
How does flow cytometry work?
As the fluorescing cells passes through the laser beam it creates a peak or pulse of photon emissions over time
These are detected by the PMT and converted to a voltage pulse, known as an event
The total pulse height and area are measured by the flow cytometer and it will correlate directly to intensity of fluorescence for that event
Each event is given a channel number depending on its measured intensity
The more intense the fluorescence, the higher the channel number assigned to the event
What’s a gating strategy?
To identify cells if interest based n size and granularity
To ignore debris or dead cells
To ignore doublets or triplets or more
