Seminar of Cell Culture Techniques - PowerPoint PPT Presentation

About This Presentation
Title:

Seminar of Cell Culture Techniques

Description:

Seminar of Cell Culture Techniques Tapodi Antal Department of Biochemistry and Medicinal Chemistry, Faculty of Medicine, University of Pecs, Hungary – PowerPoint PPT presentation

Number of Views:260
Avg rating:3.0/5.0
Slides: 56
Provided by: Cser8
Category:

less

Transcript and Presenter's Notes

Title: Seminar of Cell Culture Techniques


1
Seminar of Cell Culture Techniques
  • Tapodi Antal
  • Department of Biochemistry and Medicinal
    Chemistry, Faculty of Medicine, University of
    Pecs, Hungary

2
Contents
  • I. Cells Types
  • II. Introduction to Cell Culture Lab
  • III. Techniques

3
I. Cell Types
  • Primary cultures
  • Secondary cultures
  • Normal
  • Immortalized
  • Spontaneous Transformation
  • Transfection
  • Somatic Cell Fusion(Hybridomas, Hybrids)
  • Cell lines
  • Adherent
  • Suspension
  • Cells from ATCC and ETCC

4
1. Primery Cultures
  • Tissue preparation from young animal, or
    isolation of cells from blood, intraperitoneal
    fluid, etc.
  • Tissue dissociation
  • Dissection then Homogenization with Knife or
    Blender
  • Enzymatic Digestion (collagenase, papain,
    trypsine)/cleaving of DNA of damaged cell with
    DNase
  • Dissociation of cells in medium and selection of
    organic cell types

CO2 Incubator
Knife Blender
5
2. Secondary cultures
H9c2
  • Normal cell lines
  • They were spontaneously immortalized.(e.g.
    Cardio-myocytes from rat)
  • Immortalized
  • Transfected with some sort of oncogene SV40
    (Simian virus)Large T antigen
  • (T IDBL)
  • Tumor cells (e.g. Human cervix carcinomas HeLa)
  • Hybridomas

HeLa
6
Hybridomas
  • Cell fusion of
  • HGPRT and TK-/- myeloma and B-cells from
    immunized animal
  • Selection of hybridomas in HAT (Hypoxanthine,
    Aminopterine and Thymidine) medium

7
Hybrid selection
  • Metabolic pathways relevant to hybrid selection
    in medium containing hypoxanthine, aminopterin
    and thymidine (HAT medium).
  • When the main synthetic pathways are blocked with
    the folic acid analogue aminopterin (), the cell
    must depend on the salvage enzymes HGPRT and TK
    (thymidine kinase). HGPRT (-) cells cannot grow
    in HAT medium unless they are fused with HGPRT
    () cells.

8
Effect of HAT-medium Selection
9

5-Amino Imidazole-

4-Carboxy Ribonucleotide



5-Formido-Imidazole-

4-Carboxamine Ribo-

nucleotide
PRPP PP
Hypoxanthine
Inosine Monophosphate
Hypoxanthine Guanine
Phosphoribosyl Transferase

(HGPRT) Guanine
Guanosine
Monophosphate

(GMP) PRPP
PP Thymidine
GDP dGDP

Thymidine kinase RNA GTP
dGTP
dTMP dTDP d TTP
DNA
Thymidylate
Synthetase
UDP dUTP dUMP
dCTP dATP
10
Production of Polyclonal and Monoclonal antibodies
11
Neuro Hybryds
  • It works with adherent cells.
  • Cell fusion of HGPRT and TK-/-, no secreting
    neuoblastoma and neural cells.
  • Selection in HAT medium

12
Cell lines
  • Adherent (WRL-68, HepG2, HeLa etc.)
  • Suspension (Jurkat)
  • Cells from ATCC and ETCC

Jurkat
WRL-68
HeLa
HepG2
13
Online Order of Cell Lines
14
II. Introduction of Cell Culture Lab(Equipment)
  • CO2-thermostats
  • Airflow
  • Solutions
  • Dishes
  • Freezers
  • Liquid nitrogen
  • Centrifuges
  • Autoclave
  • Vacuum ovens
  • Cryotubes
  • Microscopes
  • ELISA-readers

15
CO2 Incubators
  • Water Jacketed CO2 incubator
  • 3 Gas/CO2 Incubator with RH Control
  • Precise control of Oxygen levels combined with
    CO2, N2 and RH ensure accurate conditions for
    applications such as, hypoxic cell studies and
    cancer research.

16
Laminar Flow Box
  • HEPA filter rated at 99.99 efficient for 0.3
    micron particulates. The HEPA filtered air is
    then directed vertically across the work surface.

17
Dishes
  • Dishes
  • Multiwell plates
  • Flasks
  • Flasks on slide

18
Freezers
19
Centrifuges
20
Autoclaves
21
Vacuum Ovens
22
Microscopes
23
ELISA readers
24
FACS
25
II. Introduction of Cell Culture Lab(Culture)
  • Growth of the cells in adequate media with serum
    (FCS/FBS) and antibiotics and antimycotics
    (chemically defined serum-free media)
  • Environment
  • Temperature 37C (34 C, 41 C)
  • High humidity
  • 5 CO2
  • Split Trypsin-EDTA
  • Count of Cells (Thrypan Blue)

26
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

27
Metabolic activity(MTT, viability assay)
4
  • Seed the cells into 96-well plates at a starting
    density of 10 cell/well and culture overnight in
    humidified 5 CO2 atmosphere at 37 C.
  • Treat the cells modifying the their viability the
    following day.
  • Remove medium from the wells containing 0,5
    water suluble mitocondrial dye,
    (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazo
    lium bromide (MTT)
  • Incubate 3 hours and solubilize the water
    insoluble blue formasan dye by 10 SDS in 10mM
    HCl
  • Determine the optical density by an ELISA reder
    at 550 nm wavelength

28
Effect of HO-3089 (Novel PARP-inhibitor) on
WRL-68 in Oxidative Stress
29
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

30
Apoptosis signalling
31
Activation and inhibition of Apoptosis
32
The Roll of mitochondria in apoptosis
33
Caspase Cascade
34
Fluorescent dyes I.
  • Hoechst 33342blue
  • Selective nuclear dye
  • Chromatin condensation, fragnentation
  • Rhodamine 110 green
  • Bis-L-asparic acide amide (substrate by caspase
    3) green
  • TMRE polarization of mitochondria red

35
Fluorescent dyes II.
  • Propidium iodide Late-stage apoptotic and
    necrotic cells red
  • YO-PRO-1 Viable cell nuclei green
  • Annexin V early-stage apoptotic cells green

36
DNA Laddering
  • To investigate the DNA fragmentation, the
    extracted DNA has to run on 1,5 agarose gel.
  • DNA fragments show ladder-pattern.

37
DNA Laddering
38
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

39
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

40
PARP(poly-ADP-rybose-polymerase)
  • Nuclear enzyme
  • Structure of PARP
  • 1st activator of PARP are ssDNA-breaks
  • The roll of PARP in necrosis and apoptosis or
    repair-mechanism
  • The roll of PARG

41
Reaction catalyzed by PARP
Ad
Ad
Ad
N

-R-P-P-R-R-P-P-R-R-P-P-R-R-P-P-R
PARP
Glu
CONH
2
Nic
42
(No Transcript)
43
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

44
Transfection I.
pEGFP with NLS
  • Expression vector systems
  • pcDNA
  • pEGFP

pEGFP without NLS
45
Transfection II.
  • RNAi
  • siRNA
  • stRNA or Dicer RNAi
  • shRNA Using vectors for RNAi analysis
  • siRNA cassette

46
Proposed mechanism for how siRNA works
47
stRNA or Dicer RNAi
48
Gene deletion (Demonstration)
49
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 produce cultures
    and Characteristics of particular tissue.
  • Possibility by damage and ageing (Parkinsons
    disease, diabetes)

50
Epithelial Stem Cell identification and isolation
  • First methods involved in the separation of an
    epithelial cell type from other cells will be
    examined, followed by ways in which the
    proliferative capacity of such a cell type can be
    assessed.
  • Secondly, methods used for the maintenance of
    primery stem cells in culture and ways of
    caracterizing stem cells using immunocytochemistry
    will be described.

51
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

52
Detection of Bladder Cancer
  • The probe was designed to detect aneuploidy for
    chromosomes 3, 7, 17 and loss of the 9p21 locus
    via fluorescence in situ hybridization (FISH) in
    urine specimens from subjects with transitional
    cell carcinoma of the bladder.

two copies of chromosome 3 (red), four copies of
chromosome 7 (green), five copies of chromosome
17 (aqua) and one copy of p16 gene (gold)
53
Flow Cytometric Methods
  • Separation of labeled cells
  • Clinical applications

54
DNA Array technique
  • Mr. Péter Jakus

55
Cell suspension by NMR
  • Dr. Zoltán Berente
Write a Comment
User Comments (0)
About PowerShow.com