Presented by-

Bishnupriya Saha
School Of Biotechnology, KIIT University
Topics I have learned here
 Fundamentals of cell culture laboratory

 Immunohistochemistry (basics)

 H & E staining

 Western blotting
Cell culture fundamentals
 What is cell culture

 Why do we need cell culture

 Primary cells, cell lines

 Confluency, passage number, split ratio

 Sub culturing, types of culture methods

Contd.
 Media preparation, serum components

 Serum free media

 Different modes of sterilization

 Contamination
 Subcultivation
 Cryopreservation
Introduction
 Cell culture is the process by which cells are grown
under controlled conditions, generally outside their
natural environment.
 Types:
1. Primary cell culture
2. Finite cell culture
3. Continuous cell line
 Population doubling time- time required for cell
number to be doubled.
Importance of cell culture
 Model systems for studying basic cell biology,
effectiveness of drug, interactions between disease
causing agents and cells.
 Toxicity of cells
 Cancer research
 Genetic engineering
 Gene therapy
Types of cells
Cntd.
Cntd.
Culture media
 Choice of media depends on the type of cell being
cultured.
 Commonly used media are DMEM, EMEM etc.
 Media is supplemented with antibiotics e.g.; penicillin,
streptomycin etc.
 Prepared media is filtered and incubated at 4 °C.
 Consists of basal salts, trace elements, growth factors,
hormones, amino acids, carbohydrates, vitamins,
metabolic precursors etc.
Media components
 1. Sodium bicarbonate and buffering:
In culture media, dissolved CO₂ is in equilibrium with
bicarbonate ions and many medium formulations take
advantage of this CO₂/bicarbonate reaction to buffer the
pH of the medium.
H₂O + CO2 H₂CO₃ H+ + HCO₃–

(Optimum pH= 7.2-7.4)

H₂O + CO₂ + NaHCO₃ H+ + Na+ + 2HCO₃–
Complete media
• A medium for an in vitro culture
that contains the supplemental
nutrients as well as the basic
nutrients to support fastidious or
mutant growth requirements.
Phenol red :
 Used to monitor the pH of the media.
 During cell growth, the medium changes its color as it
changes pH due to metabolites released by the cells.
 At low pH: red turns medium yellow.
 At high pH : red turns to medium purple.
 pH 7.4 bright red.

L- glutamine :
 Essential amino acid required for cell growth.
Non-essential amino acids:
 Alanine, asparagine, glycine, asparatic acid, glutamic
acid, proline & serine.
 Reduces the metabolic burden on the cells allowing for
an increased cellular proliferation.
Fatty acids & lipids:
 Particularly important in serum free media as they are
generally present in serum.
Trace elements:
 Zn, Cu, Se, TCA
Vitamins:
 Source : glucose
 Most commonly used: Vitamin B
Serum
 Serves as a source of carbohydrates, vitamins, amino
acids, lipids, hormones, growth factors, minerals &
trace elements.
 Buffers the culture medium.
 Inactivates the protease inhibitors.
 Increases medium viscosity.
 Conditions the growth surface of the culture vessel.
 FBS & calf bovine serum most commonly used.
Serum free media
 Presence of serum in media can lead to serious
misinterpretations in immunological studies.
 These media are generally specifically formulated to
support the culture of a single cell type and
incorporate defined quantities of purified growth
factors , lipoproteins, & other proteins, which are
otherwise provided by serum.
 Also referred to as “ defined media”.
Carbohydrates
 Major source of energy
 Glucose, galactose, maltose, fructose
Proteins & peptides
 1. Albumin: Main protein in blood acting to bind H2O,
salts, free fatty acids, hormones, vitamins and
transport them between tissues & cells.
 2.Fibronectin: Key player in cell attachment.
 3. Transferrin: Iron transport protein.
Acts to supply iron to cell membrane.
Confluency
 The state when all available space of the culture vessel
is covered due to cellular expansion.
Cntd.
Cntd.
 Cells to be kept in healthy and in growing state have to
be sub cultured or passaged, it’s the passage of cells
when they reach to 80-90% confluency in flasks/
dishes/ plates.
 Enzymes such as trypsin in combination with EDTA
breaks the cellular bond that attached the cells to the
surface.
Anchorage dependent &
independent cell lines
Anchorage dependent Anchorage Independent

 In culture vessels , when cell lines  When cell lines grow either attached
grow & divide in a monolayer or in to a surface or in suspension.
suspension.  Suspension cells are anchorage
 Cell lines derived from normal independent. e.g.; blood cells.
tissues are considered as anchorage
dependent, grows only on a suitable
substrate. e.g.; tissue cells.
Adherent cells.
Culturing of adherent cell lines
 Cells are washed with
PBS( Ca & Mg free)solution.
 Add enough trypsin/EDTA to
cover the monolayer.
 Incubate the cells in 37 C
for 1-2 minutes.
 Tap the vessels from the sides
to dislodge the cells.
 Add complete medium to
dissociate and dislodge the cells
by the help of pipette which
are remained to be adherent.
Culturing of suspension cell lines
 Easier to passage as no
need to detach them.
 As the suspension cells
reach confluency, remove
1/3rd of the medium &
replace with the same
amount of pre-warmed
medium.
Examination of culture
•For microscopic
evidence of
microbial
contamination
for ex.- pH shifts,
turbidity or
particles
Cell counting
 Necessary in order to establish or monitor growth rates
as well as to set up new cultures with known cell
numbers.
 Hemocytometers are commonly used to estimate cell
number and to determine cell viability.
Hemocytometer
Calculations
 % Cell viability: (Total viable cells/ total cells)*100

 Viable cells/ml : avg. viable cell count per square*

dilution factor* 10^4

 Total viable cells/ sample: viable cells/ml* the original

vol. of fluid from which the cell sample was removed.
Cell viability
 Viable assays measure the number of viable cells in a
population.
 Provides an accurate indication of the health of the
cell culture.
 Trypan blue and erythrosin b.
Trypsinization
 To detach adhered cells from the surface.
 Involves the breakage of both intercellular and
intracellular cell to surface bonds and digestion of
their protein attachment bonds with proteolytic
enzymes such as trypsin/EDTA.
 Not required in suspension culture.
Types of trypsinization
• Warm trypsinization is involved in the • Cold trypsinization is involved in the
incubation of tissues with warm trypsin soaking of tissues in cold trypsin at 4 °C
at 36.50 °C . followed by the incubation at 36.50 °C.

• Damage to the cells is high in warm • Cold trypsinization minimizes the

trypsinization . prolonged effect of warm trypsin on the
tissue.
 Cryopreservation
• Refers to the
preservation of biological
tissues in sub-zero
temperatures , typically -
196C.
•At these temperatures, all
biological activities of cells
is effectively stopped or
ceased.
•Provides indefinite
longevity to cells.
Procedure
Contamination
 Contamination can arise from different sources.
 1.Mycoplasma
 2.Bacteria
 3.Fungi
 4.Molds
 5.Cross contamination
Different Instruments used
 Autoclave( for sterilization)
 Biosafety cabinet
 Centrifuge
 Magnetic stirrer
 Vortex machine
 Inverted Microscope
 Refrigerator and freezer
 CO2 Incubator
 Liquid nitrogen chamber( for cryopreservation)
Expanded Instruments
 Water bath
 Hemocytometer
 Micropipettes, tips
 Glass beakers, jars, measuring cylinders
 PPE ( personal protective equipments)
 Paraffin chits
 Falcon tubes
 Weighing machine
Bio safety
cabinet Inverted microscope
CO2 Incubator Cell culture flasks
IHC( Immuno-histo-chemistry)
 IHC combines histological, immunological and
biochemical techniques for the identification of
specific tissue antigens by means of a specific antigen-
antibody reaction tagged with a visible label.

 To visualize the localization and distribution of

specific cellular components within a tissue.
Significance
 Enables the observation of processes in the context of
intact tissue.
 Useful for assessing the progression & treatment of
diseases such as cancer.
 Major changes in the expression pattern of an antigen.
 Specific cell or tissue expression of an antigen.
Principle
•The localization of
antigens in tissue
sections by the use
of labeled
antibodies as
specific reagents.
•Visualization is
done by a marker
such as fluorescent
dye, enzyme etc.
http://samdeetresearchproject.blogspot.
com/2016/04/immunohistochemistry.ht
ml
Antigen & antibody

https://microbenotes.com/differences-between-antigen-and-antibody/
Protocol
 The antibody-antigen interaction is visualized using
1. Chromogenic detection : enzyme conjugated to the
antibody- cleaves a substrate to produce a colored
precipitate at the location of the protein.

2. Fluorescent detection: a fluorophore is conjugated

to the antibody & can be visualized using
fluorescence microscopy.
Basic steps
 Biopsy
 Fixing and embedding
 Cutting and monitoring the sections
 Deparaffinization and rehydrating the sections.
 Antigen retrieval HIER
enzymatic
 IHC staining
 Counter- staining( if desired)
 Dehydrating & stabilizing with mounting medium (dpx)
 Viewing the staining under the microscope.
Basic steps

https://www.creative-bioarray.com/protocol/immunohistochemistry-
protocol.htm
H & E Staining
 Principle stain used for studying histology & medical
diagnostics.
 Basically it is used for detecting the changes in
morphology of the tissue.
 ‘H’ hematoxylin stains the nucleus BLUE

 ‘E’ eosin( acidic) stains the cytoplasm PINK

 Inexpensive
Major steps in H&E staining
 1. Deparaffinization( by xylene)
 2. Hydration( graded alcohols to water)
 3. Nuclear staining (by hematoxylin)
 4. Counterstaining (using eosin)
 5. Dehydration ( application of graded alcohol to 100%
alcohol)
 6. Clearing (xylene- transition from alcohol to non-
aqueous reagents)
 Visualization

Nucleus

Cytoplasmic
proteins
Western Blotting
 A widely used technique to monitor protein expression
in a cell/tissue extract based upon antibody binding to
a specific protein of interest.
 Antibodies used in western blotting can be specific to
total protein or to a post translationally modified
version of a protein such as sites of phosphorylation,
acetylation, methylation, ubiquitylation.
 To validate new & existing antibodies.
 Most useful in the fields of molecular biology,
biochemistry, immunogenetics etc.
Principle
 Relies on the specificity of binding between a molecule
of interest & a probe to allow detection of the molecule
of interest in a mixture of many other similar
molecules.
 Molecule of interest – Protein
 Probe- typically an antibody raised against that particular protein

 The SDS PAGE technique is prerequisite for western

blotting.
Cntd.
 This technique uses three elements to accomplish the
task:
• Separation by size
1.

• Transfer to a solid support

2.
• Making target protein using a proper
primary or secondary antibody to
3. visualize.
Solutions & reagents required
 1. PBS
 2.SDS sample buffer
 3. Cell lysis buffer/RIPA buffer.
 4. Transfer buffer
 5.TBSt
 6. Blocking buffer
 7. BSA
 8. Primary antibody dilution buffer containing BSA
 9. Pre-stained protein marker
 10. Blotting membrane( PVDF/ nitrocellulose)
Importance
 To verify the expression of a protein
 To determine the relative amount of a protein present
in different samples
 Highly sensitive
 As little as 1-5 nano gram of an average sized protein
can be detected by western blotting.
 To analyze protein-protein interactions.
Methodology
 Sample preparation:
Cells in culture removal of cells lysis by
detergents & sonication.
 SDS PAGE
 Detergents bind proteins
 Denaturation of proteins
 Load proteins on gel
 Apply current
 Proteins separate by size.
SDS PAGE set up

https://www.researchgate.net/figure/Schematic-of-SDS-Page-electrophoresis-
Polyacrylamide-two-part-gel-composed-of-a-stacking_fig21_315866219
Western blot equipments

https://www.creative-diagnostics.com/The-Basis-of-Western-Blot.htm
https://www.antibodies-online.com/resources/17/1224/western-blotting-
immunoblot-gel-electrophoresis-for-proteins/
Procedure

https://www.creative-diagnostics.com/The-Basis-of-Western-Blot.htm