s5_cellculture
TRANSCRIPT
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Seminar of Cell Culture
TechniquesTapodi Antal
Department of Biochemistry and MedicinalChemistry, Faculty of Medicine, University
of Pecs, Hungary
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Contents
I. Cells Types
II. Introduction to Cell Culture Lab III. Techniques
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I. Cell Types
Primary cultures
Secondary cultures
Normal
Immortalized Spontaneous
Transformation
Transfection
Somatic Cell Fusion(Hybridomas, Hybrids)
Cell lines
Adherent
Suspension
Cells from ATCC andETCC
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1. Primery Cultures
Tissue preparation fromyoung animal, or isolation ofcells from blood,intraperitoneal fluid, etc.
Tissue dissociation Dissection then
Homogenization with Knife orBlender
Enzymatic Digestion(collagenase, papain,trypsine)/cleaving of DNA ofdamaged cell with DNase
Dissociation of cells inmedium and selection oforganic cell types
Knife Blender
CO2 Incubator
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2. Secondary cultures
Normal cell lines
They were spontaneously
immortalized.(e.g.: Cardio-
myocytes from rat)
Immortalized Transfected with some sort
of oncogene; SV40 (Simianvirus)Large T antigen
(T IDBL) Tumor cells (e.g.: Human
cervix carcinomas: HeLa)
Hybridomas
H9c2
HeLa
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Hybridomas
Cell fusion of
HGPRT and TK-/-myeloma and B-cells
from immunized animal Selection of hybridomas
in HAT (Hypoxanthine,Aminopterine andThymidine) medium
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Metabolic pathways relevant to hybrid selection inmedium containing hypoxanthine, aminopterin andthymidine (HAT medium).
When the main synthetic pathways are blocked with thefolic acid analogue aminopterin (*), the cell mustdepend on the salvage enzymes HGPRT and TK
(thymidine kinase). HGPRT (-) cells cannot grow inHAT medium unless they are fused with HGPRT (+)cells.
Hybrid selection
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Effect of HAT-medium Selection
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5-Amino Imidazole-4-Carboxy Ribonucleotide
*
5-Formido-Imidazole-
4-Carboxamine Ribo-nucleotide
PRPP PP
Hypoxanthine Inosine MonophosphateHypoxanthine GuaninePhosphoribosyl Transferase
(HGPRT)Guanine Guanosine Monophosphate
(GMP)PRPP PP
Thymidine GDP dGDP
Thymidine kinase RNA GTP dGTP
dTMP dTDP d TTP DNA* Thymidylate
SynthetaseUDP dUTP dUMP dCTP dATP
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Production of Polyclonal and
Monoclonal antibodies
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Neuro Hybryds
It works with adherent cells.
Cell fusion ofHGPRT and TK-/-, no secretingneuoblastoma and neural cells.
Selection in HAT medium
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Cell lines
Adherent (WRL-68,HepG2, HeLa etc.)
Suspension (Jurkat)
Cells from ATCC andETCC
JurkatWRL-68
HeLa HepG2
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Online Order of Cell Lines
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II. Introduction of Cell Culture Lab
(Equipment)
CO2-thermostats
Airflow
Solutions
Dishes
Freezers
Liquid nitrogen
Centrifuges
Autoclave
Vacuum ovens
Cryotubes
Microscopes
ELISA-readers
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CO2 Incubators
Water Jacketed CO2incubator
3 Gas/CO2 Incubator
with RH Control Precise control of Oxygen
levels combined withCO2, N2 and RH ensure
accurate conditions forapplications such as,hypoxic cell studies andcancer research.
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Laminar Flow Box
HEPA filter rated at99.99% efficient for 0.3
micron particulates. TheHEPA filtered air is thendirected vertically acrossthe work surface.
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Dishes
Dishes
Multiwell plates
Flasks
Flasks on slide
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Freezers
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Centrifuges
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Autoclaves
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Vacuum Ovens
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Microscopes
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ELISA readers
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FACS
II Introd ction of Cell C lt re Lab
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II. Introduction of Cell Culture Lab
(Culture)
Growth of the cells in adequate media with serum(FCS/FBS) and antibiotics and antimycotics (chemicallydefined serum-free media)
Environment: Temperature: 37C (34 C, 41 C)
High humidity
5% CO2
Split: Trypsin-EDTA
Count of Cells (Thrypan Blue)
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III. Techniques
Metabolic activity (MTT)
Detection of Apoptosis and Necrosis
Western blot from cells
Transfection Gene deletions (Demonstration) Clinical Application of cultured Human Stem Cells
Flow Cytometric Methods
FISH-probes
DNA Array
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Metabolic activity
(MTT, viability assay)
Seed the cells into 96-well plates at a starting density of 10cell/well and culture overnight in humidified 5 % CO2atmosphere at 37 C.
Treat the cells modifying the their viability the following day.
Remove medium from the wells containing 0,5% water sulublemitocondrial dye, (3-(4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT+)
Incubate 3 hours and solubilize the water insoluble blueformasan dye by 10% SDS in 10mM HCl
Determine the optical density by an ELISA reder at 550 nmwavelength
4
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0
20
40
60
80
100
Ctrl. 0 0,1 0,5 1 2
M HO-3089
Survival(%
)
Effect of HO-3089 (Novel PARP-inhibitor) on
WRL-68 in Oxidative Stress
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Apoptosis signalling
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Activation and inhibition of
Apoptosis
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The Roll of mitochondria in
apoptosis
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Caspase Cascade
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Fluorescent dyes I.
Hoechst 33342:blue Selective nuclear dye
Chromatin condensation,fragnentation
Rhodamine 110: green
Bis-L-asparic acide amide(substrate by caspase 3):green
TMRE: polarization ofmitochondria: red
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Fluorescent dyes II.
Propidium iodide: Late-stage apoptotic andnecrotic cells: red
YO-PRO-1: Viable cellnuclei green
Annexin V: early-stageapoptotic cells: green
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To investigate the DNA
fragmentation, the extracted
DNA has to run on 1,5%agarose gel.
DNA fragments show ladder-pattern.
DNA Laddering
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DNA Laddering
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Detection of Apoptosis and Necrosis
Activity of Caspase 3 and Caspase 8
Release of Cytochrome c and AIF
Fluorescence dyes Hoechst 33342
Annexin V
Propidium iodide
Rhodamine
DNA Laddering
Induction and protection
PARP
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Induction and Protection of
Apoptosis
Induction: Hydrogen peroxide
Etoposide
Death domains: TNF, FAS, TRAIL
BAD
Protection: BCL-2 family
IAP Inhibition of PARP
HSP27,70,90
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PARP(poly-ADP-rybose-polymerase)
Nuclear enzyme
Structure of PARP
1st activator of PARP are ssDNA-breaksThe roll of PARP in necrosis and apoptosis or
repair-mechanism
The roll of PARG
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-R-P-P-R-R-P-P-R-R-P-P-R-R-P-P-R
Ad
PARP Glu
AdN
CONH2
Ad
R-P-P-R-R-P-P-R
Ad Ad
+
Nic
Nic-R-P-P-R
Ad
(NAD+)
Reaction catalyzed
by PARP
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III. Techniques
Metabolic activity (MTT)
Detection of Apoptosis and Necrosis
Western blot from cells
Transfection Gene deletions (Demonstration)
Clinical Application of cultured Human Stem Cells
FISH-probes
Flow Cytometric Methods
DNA Array
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Transfection I.
Expression vectorsystems
pcDNA
pEGFP
pEGFP with NLS
pEGFP without NLS
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Transfection II.
RNAi
siRNA
stRNA or Dicer RNAi
shRNA Using vectors for RNAi
analysis
siRNA cassette
d f
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Proposed mechanism for how siRNA
works
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stRNA or Dicer RNAi
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Gene deletion(Demonstration)
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Clinical Application of cultured Human
Stem Cells
Not only can human embryonic stem cells
be cultured in the laboratory.
But cells may be manipulated to producecultures and Characteristics of particular
tissue.
Possibility by damage and ageing(Parkinsons disease, diabetes)
E i h li l S C ll id ifi i
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Epithelial Stem Cell identification
and isolation
First methods involved in the separation of anepithelial cell type from other cells will beexamined, followed by ways in which the
proliferative capacity of such a cell type can beassessed.
Secondly, methods used for the maintenance ofprimery stem cells in culture and ways of
caracterizing stem cells usingimmunocytochemistry will be described.
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FISH
(Fluorescence in situ Hybridization)
Application of FISH-probes Prenatal, Postnatal and Preimplantation Genetics
Oncology, Cytology & Pathology
Hematological Cancer Etc.
Equipments:
Fluorescence Microscope Dye adequat filter sets
Sample and Reference DNA
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Detection of Bladder Cancer
The probe was designedto detect aneuploidy forchromosomes 3, 7, 17
and loss of the 9p21locus via fluorescence insituhybridization (FISH)in urine specimens from
subjects with transitionalcell carcinoma of thebladder.
two copies of chromosome 3 (red),four copies of chromosome 7(green), five copies of chromosome17 (aqua) and one copy of p16 gene(gold)
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Flow Cytometric Methods
Separation of labeledcells
Clinical applications
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DNA Array technique
Mr. Pter Jakus
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Cell suspension by NMR
Dr. Zoltn Berente