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Seminar of Cell Culture Techniques
Seminar of Cell Culture Techniques
Seminar of Cell Culture Techniques
Tapodi Antal Department of Biochemistry and Medicinal Chemistry, Faculty of Medicine, University of Pecs, Hungary
Contents
I. Cells Types II. Introduction to Cell Culture Lab III. Techniques
I. Cell Types
Cell lines
Normal Immortalized
Adherent Suspension
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
2. Secondary cultures
H9c2
They were spontaneously immortalized.(e.g.: Cardiomyocytes from rat) Transfected with some sort of oncogene; SV40 (Simian virus)Large T antigen (T IDBL) Tumor cells (e.g.: Human cervix carcinomas: HeLa) Hybridomas
Immortalized
HeLa
Hybridomas
Cell fusion of HGPRT and TK-/myeloma and B-cells from immunized animal Selection of hybridomas in HAT (Hypoxanthine, Aminopterine and Thymidine) medium
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.
PRPP
Hypoxanthine
PP
Inosine Monophosphate
Hypoxanthine Guanine Phosphoribosyl Transferase (HGPRT) Guanine PRPP Thymidine Thymidine kinase RNA PP GDP GTP d TTP dCTP dGDP dGTP DNA dATP Guanosine Monophosphate (GMP)
UDP
Neuro Hybryds
It works with adherent cells. Cell fusion of HGPRT and TK-/-, no secreting neuoblastoma and neural cells. Selection in HAT medium
Cell lines
Adherent (WRL-68, HepG2, HeLa etc.) Suspension (Jurkat) Cells from ATCC and ETCC
WRL-68
Jurkat
HeLa
HepG2
CO2 Incubators
Precise control of Oxygen levels combined with CO2, N2 and RH ensure accurate conditions for applications such as, hypoxic cell studies and cancer research.
HEPA filter rated at 99.99% efficient for 0.3 micron particulates. The HEPA filtered air is then directed vertically across the work surface.
Dishes
Freezers
Centrifuges
Autoclaves
Vacuum Ovens
Microscopes
ELISA readers
FACS
Growth of the cells in adequate media with serum (FCS/FBS) and antibiotics and antimycotics (chemically defined serum-free media) Environment:
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
Seed the cells into 96-well plates at a starting density of 10 4 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-diphenyltetrazolium 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
100 80
Survival (%)
Activity of Caspase 3 and Caspase 8 Release of Cytochrome c and AIF Fluorescence dyes
Apoptosis signalling
Caspase Cascade
Fluorescent dyes I.
Hoechst 33342:blue
Rhodamine 110: green Bis-L-asparic acide amide (substrate by caspase 3): green TMRE: polarization of mitochondria: red
Propidium iodide: Latestage apoptotic and necrotic cells: red YO-PRO-1: Viable cell nuclei green Annexin V: early-stage apoptotic cells: green
DNA Laddering
To
investigate the DNA fragmentation, the extracted DNA has to run on 1,5% agarose gel. DNA fragments show ladderpattern.
DNA Laddering
Activity of Caspase 3 and Caspase 8 Release of Cytochrome c and AIF Fluorescence dyes
Induction:
Protection:
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
R-P-P-R-R-P-P-R
Ad PARP Glu
Ad
Ad
N
-R-P-P-R-R-P-P-R-R-P-P-R-R-P-P-R
+
Nic
CONH2
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
Transfection I.
pEGFP with NLS
pcDNA pEGFP
pEGFP without NLS
Transfection II.
RNAi siRNA stRNA or Dicer RNAi shRNA Using vectors for RNAi analysis siRNA cassette
Gene deletion
(Demonstration)
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.
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
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)
Pter Jakus
Zoltn Berente