Application

Introduction to cells
 Hemocytometer
(or counting chamber)
A specimen slide which is
used to determine the
concentration of cells in a
liquid sample, frequently
used to determine the
concentration of blood cells
a grid (an arrangement of
squares of different sizes) is
etched into the glass of the
hemocytometerallows for
an easy counting of cells.
 Loading the
hemocytometer
Both the hemocytometer &
its coverslip should be clean.
Then place the pipette tip
with your sample into one of
the V-shaped wells & gently
expel the sample. The area
under the coverslip fills by
capillary action.
You can load two samples on
one hemocytometer
 Counting the cells
Cells that are on the
line of a grid require
special attention. Cells
that touch the top and
right lines of a square
should not be counted,
cells on the bottom
and left side should be
counted using a
microscope.
To distinguish b/w dead &
viable cells

The sample is often diluted

with a particular stain, such as
Trypan blue. This staining
method, also known as dye
exclusion staining, uses a
diazo dye that selectively
penetrates cell membranes of
dead cells, coloring them blue
& is not absorbed by
membranes of live cells. When
viewed under a microscope,
dead cells would appear as
dark blue.
 Flow cytometry

Itis alaser-based, technology employed incell

counting,cell sorting,biomarkerdetection
&protein engineering, by suspendingcellsin a
stream of fluid & passing them by an electronic
detection apparatus. It works by passing
thousands of cells per second through a laser
beam & capturing the light that emerges from
each cell.
Gathered data is statistically analyzed to
report cellular characteristics such as size,
complexity, phenotype & health
Working:
Cells are passed one by one through the laser beam.
A no. of detectorsone in line with the light beam
(Forward Scatteror FSC) , several perpendicular to it
(Side Scatter or SSC) & one or morefluorescence
detectors.
Each particle, passing through the beam scatters the
ray & fluorescent chemicals found in or attached to the
particle may beexcitedinto emitting light. This
combination ofscattered&fluorescentlight is picked up
by the detectors.
FSC correlates with thecellvolume & SSC depends on
the inner complexity of the particle. This is because the
light is scattered off of the internal components of the
cell.
 Cell fractionation
It is the separation of homogeneous sets from
a larger population of cells.
Homogenization
Tissue is typically homogenized in
anisotonicbuffer solution, as well as a pH
buffer by use of a variety of mechanisms such
as grinding, chopping, pressure changes,
osmotic shock, freeze-thawing, & ultra-sound
homogenization. This is done to stop osmotic
damage. The samples are then kept cold to
prevent enzymatic damage .
 Filtration
Commonly, filtration is achieved either
by pouring through gauze or with a
suction filter.
Purification
Invariably achieved byDifferential
centrifugation- the sequential increase
in gravitational force results in the
sequential separation of organelles
according to their density.
 Microfluidic cell culture
Cell culture is a key step in cell biology,
tissue engineering, biomedical
engineering, & pharmacokinetics for drug
development. Microfluidic systems can
provide an in vivo-like environment for a
cell culture as well as a reaction
environment for a cell-based assay.
Chemical components of cell
(Application)
 Biosensor
Biosensor is a device for the detection of an
analyte that combines a biological component
with a physicochemical detector component. It
consists of 3 parts:
(1): The ''sensitive biological element'' (biological
material (eg. tissue, , , enzymes,antibodies etc)
can be created by biological engineering.
(2): The ''transducer'' or the ''detector element''
that transforms the signal resulting from the
interaction of the analyte with the biological
element into another signal (i.e., transducers)
that can be more easily measured & quantified;
(3): Associated electronics or signal processors that
are primarily responsible for the display of the
 Avidin: Biotin Binding
Biotin
Biotin (vitamin H) is a small molecule. The
valeric acid side chain of this molecule can
be derivatized to incorporate various
reactive groups that are used to attach
biotin to other molecules. Once biotin is
attached to a molecule, the molecule can
be affinity-purified using an immobilized
version of any biotin-binding protein.
 Avidin
Avidin is a glycoprotein that shows
considerable affinity for biotin. Avidin
& other biotin-binding proteins e.g.,
streptavidin, have the ability to bind
up to four biotin molecules making
this interaction ideal for both
purification & detection strategies.
 Advantages of using Avidin-Biotin Systems
The avidin-biotin complex is the strongest
known non-covalent interaction b/w a protein &
ligand. The bond once formed, is unaffected by
extremes of pH, temperature & other
denaturing agents. These features of biotin &
avidin are useful for purifying or detecting
proteins. Applications for which the avidin-biotin
interaction is used include:
Enzyme linked immunosorbent assay (ELISA)
Western, Northern & Southern blotting
Cell-surface labeling
Affinity purification
Electromobility shift assays (EMSA)
APPLICATION
DNA & Chromosomes
 DNA-DNA hybridization

Amolecular biologytechnique that

measures the degree of genetic
similarity b/w pools ofDNAsequences
If the strands of DNA are heated, they
will separate & as they cool, the
attraction of the nucleotides will make
them bond back together again.
 To compare different species:
scientists cut the DNA of the species into
small segments.
Separate the strands & mix the DNA
together. When the two species DNA
bonds together, the match b/w the two
strands will not be perfect since there
are genetic differences b/w the species
The more imperfect the match, the
weaker the bond b/w the two strands.
These weak bonds can be broken with
just a little heat.
 Humans &
chimpanzees
carried DNA
more similar
to one
anothers
than to
orangutans
or gorillas
DNA
 DNA microarrays
A DNA ""chip"" or microarray (no bigger
than a microscope slide) is prepared on a
small solid base such as a piece of glass or
nylon divided into a grid of tiny squares.
To each square is attached a specific piece
of DNA sequence that can act as a probe
for a particular gene. A single stranded
DNA sample of interest is cut up & then
washed over the chip.
 Any sequence in the sample that matches a
sequence on the chip will hybridise to it & if
the sample is suitably labeled (usually with
a fluorescent tag) the pattern of matches
can be visualised & analysed by computer.
DNA chips have many applications e.g.,
they can be used to look for mutations in a
gene, to measure how active a set of genes
is, or to compare the gene expression
profiles of different samples like the
genotypes of people who do or do not
respond to a particular drug"