Growing Cells in Culture

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Growing Cells in Culture

• Part 4 Equipment

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CO2 incubator

• maintains CO2 level (5-10), humidity and
  temperature (37o C) to simulate in vivo
  conditions.

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Water bath

• To warm media, TRED and PBS before placing on
  cells
• Can harbor fungi and bacteria, spray all items
  with 70 ethanol before placing in the hood.
• Usually takes 10 -15 minutes for media to warm,
  5-10 for TRED to thaw

4
Vacuum pump

• For permanent aspiration of liquids (media, PBS
  and TRED).
• Use unplugged glass pasteur pipets, throw into
  sharps box when done.

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Inverted Phase Microscope

• A phase contrast microscope with objectives below
  the specimen.
• A phase plate with an annulus will aid in
  exploiting differences in refractive indices in
  different areas of the cells and surrounding
  areas, creating contrast

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Mechanics of phase microscopy
Shifting of phase by ½ a wavelength Add and
subtract amplitudes to create more contrast
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A comparison
Phase contrast microscopy Light microscopy Can be
used on living cells requires stain, thus killing
cells
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Hemacytometer

• Specialized chamber with etched grid used to
  count the number of cells in a sample.
• use of trypan blue allows differentiation between
  living and dead cells

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Using the Hemacytometer

• Remove the hemacytometer and coverslip
  (carefully) from EtOH and dry thoroughly with a
  kimwipe.
• Center coverslip on hemacytometer
• Barely fill the grid under the coverslip via the
  divet with your cell suspension.
• Count cells in ten squares (5 on each side) by
  following diagram at station.

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Looking at the grid under the phase contrast
microscope
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How the cells will appear

• Bright refractile spheres are living cells,
• Blue cells about the same size as the other cells
  are dead.
• Keep a differential count of blue vs. clear for
  viability determination.
• Sometimes there will be serum debris, and this
  will look red or blue and stringy or
  gloppy--dont count it!

These are blood cells, You will not have this many
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Count 10 squares Any 10 will do but we will
follow convention Watch for stringy,
reddish materialthose arent cells!
serum
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Top group Count cells that touch top and left
lines
DO NOT Count cells that touch bottom and right
lines
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Bottom Group
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Calculate your cells/ml

• Calculate the number of total cells in one ml of
  your suspension.
• Total cells counted x (dilution factor) x
  (10,000)
• number of squares
• Usually, dilution factor is 2 and of squares is
  10
• (our example 62/10 x2 x104 1.24 x 105)

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Determine your percent viability

• Viability is a measure how many of your cells
  survived your cell culture technique.
• of viable (living) cells x 100
• total number of cells counted
• Our example 54/62 x 100 87.09

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Calculate total of cells in original suspension

• Number of cells per ml x total mls of original
  suspension
• Lets assume 10ml original suspension
• 1.24 x105 x 10 1.24 x 106 cell total
• Total of viable cells available in original
  suspension
• Total number of cells in original suspension x
  viability
• 1.24x106 x 87 1.08x 106 viable cells in the
  original suspension

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Determine the number of cells you need to add to
your flask

• You want the cells to grow happily without
  overcrowding (or being too sparse) before the
  next time you come into class.
• Using the calculation on the next slide, figure
  out the number of cells needed for the size of
  vessel being used
• You need to take into account
• length of time cells are to be grown.
• the size of the cells (not directly in the
  formula)
• their doubling time

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Your first prelab exercise

• You will be using a T-25 flask and using cells
  that have a doubling time of 18 hours
• X is the number of cells you want by the time you
  return to passage them (right column of table,
  next slide)
• X0 is the number of cells that were seeded (we
  want to solve for this right now)
• t is the time since plating (hours until the next
  passaging)
• td is the doubling time of the cell line.

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Determine how many mls of cell suspension much to
add to your flask

• of cells needed
• cells/ml

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Determine total mls fresh media you will need
to add to dish or flask

• Use table on page ___of the lab manual to see how
  many mls will fit in your flask (or we will tell
  you).
• Volume flask will hold mls suspension to
  you plan to add

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Growing Cells in Culture

• Part 5 The protocol

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Observing cells in culture

• Check color of media
• Healthy growth usually leaves media slightly
  orange
• Too yellow means bacterial growth
• Too purple means low carbon dioxide, cells dead
• Observe cells under phase microscope
• Spread out or rounded?
• How confluent?

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What to do with growing cells

• If they are at least 70-80 confluent
• Subculture them
• Also called passing or splitting
• Remove media, remove cells, resuspend and
  transfer some to a new plate

• If they are not very confluent
• Lift and replace onto same plate
• Culture more than 4 days old for our cells
• Remove old media, lift cells from plate and
  resuspend in fresh media on same plate
• Feed them
• Culture less than 4 days old
• Remove old media and replace with fresh, warm
  media

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Brief subculturing preview

• Remove media, lift cells from plate
• Resuspend cells in fresh media
• Count cells and determine viability
• Seed new plates with appropriate of cells and
  volume of media

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Some volumes that do not need to be exactbut
follow our recommendations until you are
comfortable

• Rinsing volume of PBS
• Volume of trypsin EDTA
• Volume of media to resuspend cells
• Record how much
• Volume of cells removed for counting
• Exact of cells to be plated

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You will need to return to take care of your cells

• Thursday or Friday is an in between point before
  next week.
• First time through may require up to an hour
• If one member cannot make the return time, that
  person should work in hood tonight.
• Choose a time that will be consistent each week