Culturing (Lecture 4) Flashcards
Why is solid surface good for culturing animal cells
CAMs are given a solid surface to grow
What type of media provides nine essential amino acids and often has bacteria added to reduce contamination?
Solid surface
WHat type of media includes inorganic salts, a carbon source water and tends to be faster growing for cells?
Suspension
What type of culture environment needs control temperature, atmosphere humidity and to keep strict sterile conditions?
Solid Surface
What type of culture media needs a control temperature, can use standard laboratory conditions and be kept moderately sterile?
Ssupension
______ cells are isolated directly from tissues (skin, kidney or liver)
Primary
True or False: Primary cells have a finite life span,
True
What are the three phases of human cell growth (not embryonic stem cells)?
Phase I (initial growth) Phase II (Cell strain) Phase III (Cell Senescence)
What are the three phases of mouse cell growth?
Initial loss of growth potential
Senescence
Emergence of immortal variant (cell line)
______ is an abnormal number of chromosomes.
Anueploidy
What are the three steps to isolating organelles?
- Lyse the cells
- Separate organelles based on key characteristics
- Use isolated organelles for further study
What are 3 ways to lyse a cell?
High-speed blending
Sonication
Tissue homogenizer
True or False: Swelling cells in a hypotonic solution weakens the plasma membrane, making it easier to rupture.
True
______ allows molecules to freely across the plasma membrane, along their concentration gradient.
Simple diffusion
______ (channels, carriers) allows molecules to move through a membrane protein, along their concentration gradient.
Facilitated diffusion
After lysis, mix of suspended cellular components is the ______.
Homogenate
True or False: The homogenate can be kept at room temperature.
False, kept a cool temperature so enzymes are not denatured by proteases
______ techniques separate particles based on mass or density.
Centrifugation
______ of a cell homogenate yields fractions of organelles that differ in mass and density.
Sequential differential centrifugation
______ separates cellular components by density.
Equilibrium density-gradient centrifugation
True or False: Density-gradient centrifugation is the most common initial step in protein or organelle purification.
False, differential centrifugation
How can you purify a sample of organelles?
Antibody/organelle complex can be coated on metallic beads, and then pelleted with a magnet
Antibody/organelle complex can be absorbed to killed bacteria and then pelleted by centrifugation
True or False: Organelle specific antibodies can recognize soluble proteins inside the vesicle.
False
What are the three steps of proteomics analysis of organelles?
- Isolation of organelle at high purity
- Method to identify all proteins in the organelle
Digest with protease-like trypsin (cuts at Lys & Arg)
Determine mass and sequence of peptides using mass
spectrometry to generate a ‘fingerprint’ - Genome sequence for reference comparison
Provides ‘list’ of all possible proteins
Mitochondria, chloroplast etc have their own separate genome that would need to be referenced
How can you tell which molecule is mitochondria based on proteomics.
Organelles have recognizable structure and contain characteristic proteins to perform its function
Therefore there would be a presence of ATP synthase
True or False: The best markers are the ones found in the organelle of interest and not found in any other organelle compartments.
True
