Assignments

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Assignments

• online quiz
• First lab write-up due on February 26 5pm
• Pre-lab
• Sonicator exercise

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Part 1

• Freezing cultured cells

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Reasons for freezing stocks

• Protection of valuable resource
• Cell lines in continuous culture are prone to
  variation
• Finite cell lines may become senescent
• Continuous cell lines may prone to genetic
  instability
• Contamination by microorganisms

• Cross contamination by other cell lines
• Incubator failure
• Saving time and materials maintaining lines not
  in immediate use
• Need for distribution to others

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Storage and freezing rate

• Liquid nitrogen is the most satisfactory method
  of preserving cultures
• Little deterioration of culture found at -196oC
• Significant deterioration may occur at -80oC
• Loss of 5-10 of cells per year
• Freeze slowly, preferably at 1oC/minute
• In styrofoam container, wrapped in paper
• -20oC, 1 day
• -80oC, 1 day
• Transfer to liquid nitrogen in less than 2
  minutes

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Other factors to consider

• Freeze in higher concentration of serum
• 20 rather than 10
• Freeze at high concentration of cells
• 1 x 106 or higher (up to 1 x 107)
• Freeze in presence of preservative
• Glycerol (bacterial stocks)
• Dimethyl sulfoxide DMSO (most cell lines)
• Usually at 10
• Can penetrate many synthetic and natural
  membranes, including skin
• Any potentially harmful substance can be carried
  into the circulation through the skin.
• Handle with care in the presence of any toxic
  substances

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Protocol

• Check the culture
• Healthy growth
• Freedom from contamination
• Cells should be in log phase
• Trypsinize, resuspend and count the cells
• Centrifuge resuspension and resuspend the cells
  in freezing media at 1 x 106-1 x 107 cells/ml
• Place in labeled ampules
• Date, concentration of cells, cell line (3T3-L1)
  and your initials or group number

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Centrifugation

• To increase concentration of cells
• To wash off a reagent
• Cells sediment at 80-100g
• Higher gravity may cause damage or promote
  agglutination
• MUST balance your tubes!
• Use an even number of tubes directly across from
  each other
• Either a tube of water with same mls or wait
  for another group

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Resuspension hints

• After centrifugation, will end up with small
  pellet at bottom or side of tube
• Remove all media from tube using pipettor (NOT
  Vacuum aspirator) without disturbing pellet
• Add about 1-2mls of needed freezing media to the
  tube to release pellet from side of tube and
  initially resuspend
• Add remaining freezing media to suspension in tube

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Part 2

• Lysing cultured cells

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Purpose of creating a lysate

• Liberate DNA, RNA or proteins from a cell
• Useful for research and forensic purposes
• We will use our lysate to look for proteins that
  our differentiated cells produce and the
  non-differentiated cells dont.

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How to create a lysate

• Mechanically destroy all plasma membranes and
  liberate proteins
• Outer membranes and organelle membranes
• Destroy membranes by scraping cells and
  sonication
• Also destroyed are lysosomal membranes, releasing
  the cells digestive system enzymes.
• These will destroy our proteins unless we slow
  down the enzyme function

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Slowing protein destruction

• 1 rule Keep everything cold!
• Tubes, scrapers, PBS, serum free media, plateson
  ice!
• Keep your plates on ice block and tubes in cooler
• Some researchers use PMSF
• Phenylmethylsulfonyl fluoride
• Powerful protease inhibitor
• Extremely hazardous!
• We wont be using this for this semester

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Using SDS-PAGE sample buffer with bME to help
with cell lysis

• Sodium Dodecyl Sulfate
• A detergent
• breaks membranes
• linearize the proteins
• Gives them a net negative charge
• Beta Mercaptoethanol
• Breaks disulfide bonds to linearize proteins
• Smelly!
• Bromophenol blue
• Tracking dye
• Glycerol
• Gives sample weight for loading in gel next week

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Scraping and Sonication

• Cell scraping
• Mechanical force to remove cells from dish when
  cell integrity does not need to be preserved
• Sonication
• Disrupts cell membranes with high frequency sound
  waves

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Cautions with sonication

• Pulse sample to avoid overheating
• Keep sample on ice
• Watch for overfoaming
• centrifuge afterwards
• Rinse sonicator tip in ethanol between samples
• To avoid contaminating next sample
• Dont touch tip to anything other than sample
• Includes table top, hand, etc.

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Samples to lyse

• Fibroblasts6cm dish
• Adipocytes---well plates
• Will be looking for adipsin over the next two
  weeks
• E63 3 day---6cm dish---myoblasts
• E63 7 day---6cm dish---myotubes
• Will be looking for myosin heavy chain over the
  next two weeks

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Color-coding

• Pink tube, pink label, Ffibroblast
• Green tube, green label, Aadipocytes
• Blue tube, blue label, B---myoblast lysate (3day)
• Yellow tube, yellow label, T---myotube lysate
  (7day)
• Colored tubes help you find the tubes when in ice.

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Good Technique

• Working quickly, carefully and keeping everything
  cold will ensure good results over the next two
  weeks!!
• Proteases will chop proteins up into fragments
  that are not distinctly recognizable as an entire
  protein by the antibodies in our Western blot

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Example of a good gel when lysates are prepared
properly

• Can see smears of proteins
• Proteins come in the full spectrum of sizes
• There are distinct bands embedded within
• Representing large quantities of intact proteins
• Lack of distinct bands represents poor lysate
  technique where the proteases began chewing up
  the proteins
• Most important Keep everything on ice!!!

Curving of bands in side wells is a fairly
common artifact
Pale lanes for undifferentiated cell lysates is
to be expected
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Blot of myoblast/myotubes 3/16/04 with MF20 and
Goat anti-mouse with AP

• Molecular weight standard Group 1, Group 2
  Group 3 Group 4

Excellent example of what a crisp band should
look like
First three samples show some degradation of
myosin, broken into pieces of different sizes,
but still recognizable to the antibody
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Epithelial cells

• CHO-K1
• Chinese hamster ovary
• Developed by Puck in 1957
• MDCK
• Madin Darby canine kidney epithelium
• Supports wide range of animal viruses
• ISHIKAWA
• Endometrial carcinoma from a 39 year old woman
• Estrogen and progesterone receptors demonstrated
  on the cells
• Respond to steroid hormones
• HeLa
• Human cervix epithelia
• Derived from a cervical carcinoma of a 31 yr. old
  Negro
• First aneuploid line derived from human tissue
  maintained in cell culture

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Blood

• HL60
• Peripheral blood lymphocytes obtained by
  lymphoresis from 36 year old female
• 10 will spontaneously differentiate
• Can be enhanced by using DMSO, hypoxanthine, TPA,
  retinoic acid
• THP1
• Human blood monocyte
• From peripheral blood of 1 year old male with
  acute monocytic leukemia
• Can be differentiated into macrophage-like cells
  using DMSO
• Ku-812
• From human leukemia
• Cells morphologically characteristic of basophils

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Fibroblast cells

• BHK
• Baby hamster kidney from 5 one day old unsexed
  hamster kidneys
• Good for virus replication studies
• Polio, rabies, foot and mouth disease
• NIH 3T3
• From an NIH Swiss mouse embyro
• Sensitive to leukemia virus propagation
• VERO
• Monkey kidney fibroblast line
• Also good for virus replication
• Suitable for vaccine production
• COS-1 and COS-7
• From the african green monkey kidney
• Transformed with mutant of SV40 virus
• Great transfection host

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Neuronal cells

• PC-12
• Derived from rat adrenal phaeochromocytoma
• Respond reversibly to nerve growth factors by
  induction of neuronal phenotypes
• Synthesize, store catecholamine, dopamine and NPP
• On collagen-produce neuronal shapes, without
  collagen, form spherical clusters
• SH-SY5Y
• Thrice cloned sub-line of bone marrow
  biopsy-derived line
• Can convert glutamate to neurotransmitter GABA
• Loss of neuronal characteristics with increasing
  passage numbers
• Dont use after passage 20
• Verify specific characteristic such as
  noradrenalin uptake

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Muscle cells

• C2C12
• Subclone from myoblast line established from
  normal adult C3H mouse leg muscle.
• Differentiates rapidly produces extensive
  contracting myotubes expressing characteristic
  muscle proteins.
• Provides model to study in vitro myogenesis and
  cell differentiation.
• L6-G8
• A subclone of L6, a rat thigh muscle cell line.
• Previously, on reaching confluence the cells
  fused to form myotubes and a twitching movement
  could be observed.
• However, these cells have lost the ability to
  fuse after reaching confluency.

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Where to get cell lines

• ATCC
• American type culture collection
• ECACC/Sigma Aldrich
• European Collection of Cell Culture

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Media, an incomplete list

• MEM
• Minimum Essential Media
• Sometimes called Eagles MEM or Basal Medium
  Eagle (BME)
• First widely used media, supports a broad
  spectrum of mammalian cells
• Developed by Harry Eagle (1950s)
• DMEM
• Dulbeccos Modified Eagle Media
• 2x the amino acids and 4x the vitamins of MEM
• Most often contains 4.5mg/L glucose
• Standard medium for mammalian cells

• Hams F12 (Nutrient Mixtures)
• Complex formula suitable for serum free
  propagation
• DMEM and F12 can be mixed together to support a
  broad range of cells
• First developed to support CHO cells

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More Media

• L15 Leibovitz
• Can be used without CO2 incubation (caps of
  flasks can stay tightly closed!)
• Found in teaching laboratories or when collecting
  biopsy samples
• Standard sodium bicarbonate/CO2 buffering system
  is replaced by
• Combo of phosphate buffers (Hanks balanced salt
  solution)
• Free-base amino acids
• Higher levels of sodium pyruvate and galactose

• RPMI and McCoys
• Developed at Roswell Park Memorial Institute in
  Buffalo New York.
• McCoys was originally used to grow hepatoma
  cells and supports growth of primary cultures
• RPMI is modified McCoys
• Long term culture of peripheral blood lymphocytes
• Suspension or monolayers
• Suitable for a wide variety of suspension
  cultures

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Even more media

• Glasgow minimum essential media (G-MEM)
• Developed for the culture of baby hamster kidney
  cells (BHK-21)
• Neurobasal media
• Specially designed to support neurons

• Iscoves modified Dulbeccos media
• Suitable for fast growing, high density cell
  cultures
• Highly enriched synthetic media
• Medium 199
• Popular for fibroblast cultures
• Originally formulated for chick embryo
  fibroblasts

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Balanced Salt Solutions

• PBS
• Earles buffered salts solution (EBSS)
• Higher bicarbonate concentration compatible with
  growth in 5 CO2
• Hanks buffered salts solution (HBSS)
• Good for sealed flasks with a gas phase of air

• Inorganic salts and may include sodium
  bicarbonate and sometimes glucose
• Diluent for amino acid concentrates and vitamins
• Isotonic wash or dissection medium

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Media Buffers, a short list

• Vary in their pKa values and toxicity to cells
• Sodium bicarbonate
• Most common and most liquid medias are prepared
  with this.
• HEPES
• 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
  acid
• Can be toxic for differentiated cell types
• Can increase sensitivity of media to phototoxic
  effects induced by exposure to fluorescent light
• Bufferall
• Contains 3 biological buffers
• Very good a reducing pH fluctuations