Microplate Reader

YUANDANI

Universitas Sumatera Utara

INTRODUCTION
• The microplate reader
a multimodal instrument that allows for a variety of experiments
to be performed and measured simultaneously.
• Microplate readers:
▫ Absorbance measurements
▫ Fluorescence measurements
▫ luminescence measurements.
• Multiwell plates are integral to the microplate reader and allow
for many experiments to be performed at once.
ANALYSIS
• Standard Curve

▫ This curve uses samples of known

concentration to generate a line of best
fit or standard curve.
▫ Experimental values are then
extrapolated to the curve or are
calculated using the equation from the
linear regression.

• Mapping the plate:

▫ standards
▫ samples
▫ blank
▫ positive
▫ negative controls
Application
• Multiplate readers are used to quantify:
▫ Cell viability
▫ Cell proliferation
▫ Protein
▫ Various metabolic processes such as reactive
oxygen species
Types of microplate readers
by wavelength
by detection
selection
technologies
mechanism

dedicated
microplate filters
readers

multimode
microplate monochromator
readers

no wavelength
selection.
Microplate Readers by detection technologies

• Dedicated Microplate Readers

▫ Dedicated microplate readers are able to measure one

single technology only either
absorbance, luminescence or fluorescence.

▫ They lack the flexibility of multimode microplate readers

▫ They often offer better sensitivity and reliability than

multimode microplate readers, and they are usually
cheaper.

▫ For example, the microplate luminometer

Microplate Readers by detection technologies

• Multimode Microplate Readers

▫ Multimode Microplate Readers are able to use more than one detection
technology.

▫ Multimode microplate readers have been traditionally expensive, but some

instruments offer a high modularity that allows the instrument to be
configured only with the options that are really needed.

▫ Multi-mode reader with up to seven detection modes.

 Common detection modes:
 absorbance
 fluorescence intensity,
 luminescence
 time resolved fluorescence
 fluorescence polarization
 nephelometry
Microplate Readers by wavelength selection
• Filters

▫ Affordable microplate readers use

normally filters to select the desired
wavelength for any given detection
method.

▫ Being the affordable option does not

mean it is the worst one: filters have a
very high transmittance and this brings
a higher sensitivity in many
applications.

▫ In addition, wavelength switching is

very fast,

▫ The main disadvantage is that you need

a different filter for each wavelenght
needed, and that reduces flexibility and
makes it impossible to perform a
wavelength scan.
 Microplate Readers by detection technologies

• Monochromator

▫ monochromators provide total

flexibility in wavelength
selection

▫ sensitivity in most applications

is worse than when using
filters.

▫ Some microplate readers can

be configured with both filters
and monochromators,
Microplate Readers by detection technologies

• No wavelength selection

▫ Luminometers usually lack filters or any other way

of wavelength selection
▫ light of all wavelengths can potentially reach the
photomultiplier.
Microplate
Microplate
❑ Flat bottomed plates are the most commonly used microplate in cell culture.

❑ Round-bottomed ones are useful when cell-cell interactions are important, as

the allow a loose cell pellet to form at the bottom of the well. Examples are T-
cell/antigen presenting cell interactions in T-cell cloning, or T-cell mediated
cytotoxicity assays.
❑U-bottom: Round-shaped well bottom. Wells with a U-bottom have no
edges, so they are well suited for stirring and washing of samples.
This bottom shape is used for agglutination and other assays requiring these
tasks.

❑ V-well plates are useful when combined with a plate centrifuge for obtaining
tight cell pellets. This permits resuspension and washing of multiple cell cultures.

❑V-bottom: Conically-shaped well bottom. Sample recovery is maximized

with the V-bottom shape.
EXAMPLES
EXAMPLES
Data analysis