PřF:Bi6725 Adv. Methods Cell Biol. - Course Information

Bi6725 Advanced Methods in Cell Biology

Extent and Intensity

0/2/0. 2 credit(s) (plus extra credits for completion). Type of Completion: graded credit.

Teacher(s)

prof. RNDr. Alois Kozubík, CSc. (seminar tutor)
prof. RNDr. Jiřina Hofmanová, CSc. (seminar tutor)
Mgr. Jiřina Medalová, Ph.D. (seminar tutor)
Mgr. Karel Souček, Ph.D. (seminar tutor)
prof. RNDr. Jan Vondráček, Ph.D. (seminar tutor)
doc. RNDr. Jan Hejátko, Ph.D. (seminar tutor)
prof. Mgr. Lukáš Žídek, Ph.D. (seminar tutor)

Guaranteed by

prof. RNDr. Alois Kozubík, CSc.
Department of Experimental Biology – Biology Section – Faculty of Science
Contact Person: prof. RNDr. Alois Kozubík, CSc.
Supplier department: Department of Experimental Biology – Biology Section – Faculty of Science

Timetable

Mon 18. 9. to Fri 15. 12. Wed 15:00–16:50 D36/215

Prerequisites

Bi3030 Animal physiology
The subject follows and belongs to the logical unit of practical courses of Animal Physiology and Special Methods of Animal Physiology.

Course Enrolment Limitations

The course is only offered to the students of the study fields the course is directly associated with.

fields of study / plans the course is directly associated with

there are 9 fields of study the course is directly associated with, display

Course objectives

At the end of the course students should be able to: perform both the classical and modern techniques used in basic and applied research (see Syllabus); use up-to-date trends and modern techniques used in basic research of molecules, specific cells and cell populations; evolve their laboratory skills; decide what is the most appropriate assay with newly obtained methodological background; work in well-equipped facilities after becoming familiar with the one of Institute of Biophysics of Academy of Sciences of the Czech Republic, v.v.i.; use obtained knowledge and skills in experiments designed for their bachelor and diploma thesis

Syllabus

• 1. Cytokinetic parameters of cells (prof. Hofmanová, BFU AV ČR, v.v.i.) Theory: Cultivation and use of cell cultures, types of plastic for cell cultures, work safety, operation of flow box, cell splitting, cytokinetic parameters (proliferation, differentiation, apoptosis) and methods of their detection. Experiments: Assessment of cell number and viability by CASY counter, assessment of level of differentiation by ALP method, detection of apoptotic cells by DAPI. 2. Mass spectrometry and separation techniques (doc. Cigánek, VuVeL) Theory: Separation techniques, liquid chromatography, HPLC, mass spectrometry, combination with separation techniques, suitable analytes, principles of sample ionisation, types of analysators. Practical demonstration: GC/MS and its use for biological application, LC/MS-MS and its use for metabolomics and use of LC/HybridMS in lipidomics. 3. Isolation of RNA (doc. Vondráček, BFU AV ČR, v.v.i.) Theory: Reasons for RNA isolation, RNases and protection against their action, isolation of RNA by organic extraction, purification of RNA involving its affinity to the columns, isolation of mRNA using the isolation kit, assessment of concentration, purity, and stability of isolated RNA. Experiments: Cells of hepatocellular carcinoma HepG2 treated and untreated by 10 nM TCDD for 24 h, isolation of total RNA by columnar kit. 4. Flow cytometry (Dr. Souček, BFU AV ČR, v.v.i.) Theory: Principles of flow cytometry and fluorescent signal detection, use for different particles, preparation of material, sorting of cells, multicolor analyses, analysis of cell cycle, fluorescent proteins. Practical demonstration: Separation and following sorting of live and dead cells according to the permeability of propidium iodide through cytoplasmic membrane. 5. Transfection (Dr. Medalová, FIŽ, MU) Theory: Principles of transfection, favorite vectors, stabile vs. transient transfection, favorite methods of transfection, their pros and cons, specific methods of transfection, spectrum of use of transfection techniques. Principles of confocal microscopy and advantages of its use comparing to the standard fluorescent microscopy. Experiments: Transfection of HEK293 cells by plasmid containing eGFP protein by the most used methods (cationic polymers, lipofection, fugene, electroporation - nucleofecion) and assessment of effectivity of used method by fluorescent microscope and flow cytometry. Practical demonstration of picture acquirement by confocal microscope including optimalization of obtained results. 6. Nuclear magnetic resonance (NMR) (doc. Žídek, CEITEC) Theory: Principles of NMR – interaction of nucleus with non-zero spin in magnetic field, magnetic field in molecules and their interaction with other nuclei and electrons, use of these interactions and relations for NMR, quantification of results by use of known distances of protons, samples suitable for NMR, use of NMR for analysis of molecular structure. Practical demonstration: Construction of NMR spectrometer, setting of basic experiments, data analysis and demonstration of spectra. 7. Quantitative real time PCR (qRT-PCR) (Dr. Medalová, FIŽ, MU) Theory: Reverse transcription of mRNA to cDNA, multiplication of cDNA by real time cycler, methods of quantification of product by fluorescence recorded after each cycle, types of qRT-PCR and their advantages, verification of PCR specifity by melting curve, ways of results evaluation, counting and ratios for concentration of solutions assessment and dilution of cellular suspenses, principles of cryostatic cutting and preservation of tissue, advantages of this method comparing to the standard paraffin samples. Experiments: Transcription of total RNA isolated in lection 3 to cDNA. Obtained cDNA is used for quantification of expression of genes for protein transporters belonging to the ABC family (ABC A1, ABC B1, ABC C1, ABC G2) and house-keeping gene HPRT by LightCycler and incorporation of fluorescent marker SYBR green. Cutting of frozen samples by cryostat. 8. Gene engineering in plants (doc. Hejátko, CEITEC) Theory: Structure gene, regulation of transcription, methods of quantitative and qualitative analysis of gene expression, DNA a protein chips, next generation transcriptional profiling, preparation of translational and transcriptional fusion of analyzed genes with reporter gene, identification of mutant by the changes of expression profiles by internet databases.

Literature

• Cell culture and upstream processing. Edited by M. Butler. 1st ed. New York: Taylor & Francis, 2007, xiv, 187. ISBN 9780415399692. info
• Mammalian cell culture : essential techniques. Edited by A. Doyle - J. B. Griffiths. Chichester: John Wiley & Sons, 1997, xv, 173. ISBN 0471970573. info
• Cancer cell culture : methods and protocols. Edited by Simon P. Langdon. Totowa, N.J.: Humana Press, 2004, xii, 360. ISBN 1588290794. info
• Animal cell culture :a practical approach. Edited by R. Ian Freshney. 2nd ed. Oxford: Oxford University Press, 1992, xix, 329 s. ISBN 0-19-963213-8. info
• Cell biological appplications of confocal microscopy. Edited by Brian Matsumoto. 2nd ed. Amsterdam: Academic Press, 2002, xii, 507. ISBN 0125804458. info
• Molecular cloning, A laboratory manual, Vol. 1-3, Eds. J. Sambrook and D. W. Russell, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 2001
• Short protocols in molecular biology, Vol. 1 and 2, 5th edition, John Wiley and Sons, Inc., 2002
• Current protocols in cytometry, Vol. 1 and 2, Ed. J. P. Robinson, John Wiley and Sons, Inc., 1997
• Methods in Cell Biology, Vol. 75, Cytometry, 4th edition, New developments, Eds. Z. Darzynkiewicz, M. Roederer, H. J. Tanke, Elsevier Academic Press, 2004
• CROWTHER, John R. The ELISA guidebook. Totowa, N.J.: Humana Press, 2001, xi, 421. ISBN 0896037282. info

Teaching methods

Short theoretic lectures followed by practical course of selected specific methods; demonstrative introduction to advanced methodologies; homeworks; class discussion with skilled teacher.

Assessment methods

During the course students must complete given protocols with filled results and answers. Course is finished by written test going through the methods demonstrated. Test contains usually 20 questions and at least 60% of correct answers is necessary to pass.

Language of instruction

Czech

Follow-Up Courses

• Bi5611c Special methods of animal physiology

Further comments (probably available only in Czech)

Study Materials
The course is taught annually.
Information on course enrolment limitations: Na předmět se vztahuje povinnost registrace, bez registrace může být znemožněn zápis předmětu!

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