Labs Flashcards
(37 cards)
What is PBS and how do we make it
phosphate buffered saline
one of the most used buffers in bioengineering
->similar tonicity to tissues, and cytoplasmic (cellular solution conditions)
-> avoid osmotic shock and any potential cell/tissue damage.
prepare a PBS solution using the
- weak acid salt (monosodium phosphate) and its conjugate base (disodium phosphate)
- adjusted to a final pH value to 7.4 using hydrochloric acid (HCl) or sodium hydroxide (NaOH).
NaCl, Na2PO4, KH2PO4, KCl
How does a pH meter work
measuring the hydrogen ion concentration in a solution (pH, defined as the negative logarithm of the hydrogen ion concentration)
This glass electrode is sensitive to hydrogen ions and generates a voltage that varies with the pH of the solution it’s in contact with. This voltage is compared to a reference voltage from a reference electrode, usually filled with a stable electrolyte solution like 3M KCl.
Decellularized plant scaffolds characteristics, advantages and challenges
Characteristics of Decellularized Plant Scaffolds:
- Physical resemblance to human organs.
- High superficial surface area.
- Excellent water transport and retention capabilities.
- Pre-existing vascular networks.
- Interconnected porosity.
- Range of mechanical properties.
Advantages of Plant-Derived Scaffolds:
- Unique and superior biocompatibility.
- Low production costs.
- Free from ethical concerns and supply limitations.
- Suitable for large-scale production and research.
Challenges with Decellularized Plant Scaffolds:
- Immature decellularization standards and methods.
- Limited research on the biocompatibility of plant extracellular matrix (ECM).( early stages of research)
scCO2 decell
supercritical carbon dioxide (scCO2) decellularization involves using scCO2, a fluid state of carbon dioxide where it behaves as both a gas and a liquid, to remove cellular material from plant leaves
Hexane (removes non-polar to go faster and more thorough) with PBS to stabilize and rinse, place in cylinder to dissolve with scCO2 and paracetic acid to sterilize.
bleach decell
bleach (remove proteins) and an alkaline agent like NaOH (saponification) heated and mixed with tissue
After, plant material is washed with PBS to remove any residual chemicals before lyophilization (freeze drying for storage) and storage.
Picogreen assay
The PicoGreen assay is a fluorescence-based quantification method for double-stranded DNA (dsDNA). It uses a PicoGreen dsDNA quantitation reagent that selectively binds to dsDNA. When bound, the PicoGreen dye becomes highly fluorescent and the intensity of this fluorescence is proportional to the amount of dsDNA present in the sample. Set the microplate reader to specific wavelength to excite it and to absorbs from its subsequent emission.
Use standard DNA tube with PicoGreen dye to measure and create a standard surve.
BioDrop assay
a BioDrop spectrophotometer quantifies DNA by measuring the UV absorbance at 260 nm, which is then used to calculate the concentration of DNA in the sample based on the known absorbance properties of nucleic acids.
we used 2uL
What is UView dye for
UView dye is a nucleic acid stain used in agarose gel electrophoresis. It is designed to intercalate with DNA and RNA, allowing you to visualize the nucleic acid bands under ultraviolet (UV) light after electrophoresis.
How does gel electrophoresis work basics
DNA is negatively charged due to the phosphate groups in its backbone, which carry a negative charge at neutral and alkaline pH levels commonly used in gel electrophoresis. This negative charge causes DNA to migrate towards the positive electrode (anode) during electrophoresis.
all have a similar charge-to-mass ratio because the negative charge is uniformly distributed along the phosphate backbone of the DNA.
primary factor determining the rate of migration through the gel is the size (length) of the DNA molecules, rather than differences in charge.
(Larger DNA fragments have more nucleotides, so they carry more overall negative charge. This means they are subject to a greater total electrical force in an electric field.Greater Mass Slows Movement: However, these larger DNA molecules also have more mass, which makes them move more slowly through the gel matrix. )
Smaller DNA fragments move through the gel matrix more easily and therefore travel further than larger ones in a given amount of time.
What are hydrogels and how are they typically composed?
Hydrogels are polymers that are cross-linked and highly swollen in water. They are often comprised of naturally occurring polymers.
Insoluble networks containing large amounts of water
What are examples of a hydrogel in the human body, and what are their properties?
Skin is an example of a hydrogel in the body. It is strong, resilient, and has extraordinary healing capabilities, despite being primarily made of water.
Cartilage is a hydrogel with astounding mechanical properties, like strength and elasticity, capable of handling large extensions and high loads in bone joints.
What are some potential uses of synthetic hydrogels?What is a common issue with many synthetic hydrogels?
Synthetic hydrogels can be used in drug delivery, as extracellular matrix mimics, and in tissue engineering.
Many synthetic hydrogels have poor mechanical properties, undergoing irreversible damage upon high loadings.
What are two essential attributes for hydrogels used in tissue engineering and how can hydrogels achieve these?
Current situation?
Hydrogels for tissue engineering need to adhere to cells and exhibit compatibility with their surroundings, as well as have mechanical strength.
Achieved by combining two different polymeric networks: one for biocompatibility and the other for mechanical strength.
bulk polymers (synthetic elastomers) are much stronger than hydrogels and hydrogels are weaker than cartilage
What does the sol-gel transition temperature indicate in gelatin?
The transition temperature marks when gelatin molecules reform from random coils into triple-helices acting as physical cross-links below 35°C; above this temperature, gelatin behaves as solutes in solution.
What is vial inversion gel for
Visualize sol-gel transition in gelatin
What happens to gelatin’s complex viscosity during the sol-gel transition?
The complex viscosity of gelatin increases as it transitions from liquid (sol) to gel over time.
What is alginate and how does it work
Alginate (or alginic acid) is a naturally occurring polymer found in the cell walls of brown algae. It is water soluble, wherein the cells it binds with water to form a viscous sticky gum. It is technically a copolymer, with blocks (segments of repeating units) of both ß-D- mannuronate (M) and its C-5 aptamer α-L-guluronate (G).
How does alginate cross-linking work
Crosslinking involves using divalent calcium ions (Ca^2+) to form non-covalent bonds between alginate polymer chains. The calcium ions can bind reversibly with the acid groups in the alginate’s G units, creating interchain bonds. This process replaces the monovalent sodium ions (Na^+) in the alginate, which can’t act as crosslinkers, with calcium ions that can form bonds between multiple chains, thus creating a crosslinked network.
Cross-linking polymers
Crosslinking polymers refers to the process of chemically or physically connecting the polymer chains together, creating a network structure. This crosslinking enhances the strength, stability, and various properties of the polymer, such as its elasticity and resistance to heat and chemicals.
What types of materials do we have in a stress strain sense
brittle material, breaking at low strain, but requiring a relatively high force (or stress). (steep slope)
tough material, exhibiting strength but also relatively high elongation. (steep with plateau)
soft material, being highly extensible, but breaking with relatively little force. (not v sleep, low plateau)
What is stress and strain, curve
stress = sigma = F/A0
Strain = lambda = L stretched / L original
How does mycoplasma detection work
researchers design PCR primers that are complementary to mycoplasma DNA, so when mycoplasma is present in the sample, the PCR amplification will produce a product of the expected size, which is typically around 500 bp. This is a common strategy for detecting mycoplasma contamination because it provides a specific and reliable way to identify its presence in cell cultures.
How does sds quantification work
In our SDS quantification protocol, a standard curve is constructed using diluted SDS solutions, and then samples containing SDS are mixed with Methylene Blue and Chloroform (solubilize sds and methylene), incubated, and their absorbance at 650 nm is measured using a spectrophotometer. The concentration of SDS in the samples is determined by comparing their absorbance to the standard curve, allowing for quantification of SDS content.
the dye and sds form a complex which changes the absorbance
scCO2 mammalian
osmotic shock, trypsin for protein wash, PBS,
scCO2 for decell
wash again
scCO2 for sterilization
