Bi6725 Advanced Methods in Cell Biology
Faculty of Science
Autumn 2011 - acreditation
The information about the term Autumn 2011 - acreditation is not made public
- Extent and Intensity
- 0/2/0. 2 credit(s) (plus extra credits for completion). Type of Completion: graded credit.
- prof. RNDr. Alois Kozubík, CSc. (seminar tutor)
prof. RNDr. Jiřina Hofmanová, CSc. (seminar tutor)
Mgr. Jiřina Procházková, Ph.D. (seminar tutor)
Mgr. Karel Souček, Ph.D. (seminar tutor)
doc. RNDr. Jan Vondráček, Ph.D. (seminar tutor)
- prof. RNDr. Alois Kozubík, CSc.
Department of Experimental Biology - Biology Section - Faculty of Science
Contact Person: prof. RNDr. Alois Kozubík, CSc.
- The subject follows and belongs to the logical unit of practical courses of Animal Physiology and Special Methods of Animal Physiology.
- Course Enrollment Limitations
- The course is only offered to the students of the study fields the course is directly associated with.
- Fields of study the course is directly associated with
- there are 6 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 methodic background;
ork 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
- 1. Cell cultures
- Assessment of cell count and viability: laboratory equipment, work in the sterile flow box, sorts of plastic for cell culture, safety rules in molecular biology labs, passaging of cells, counting of cells with use of Coulter Counter, assessment of cells viability by eosin and propidium iodide dye exclusion assays.
- Detection of apoptotic cells: assessment of apoptotic index - DAPI staining (detection of fluorescent cells with condensed and fragmented chromatin). Measurement of mitochondrial membrane potential of apoptotic cells by TMRE (tetramethyl rhodamin ethylester) by flow cytometry.
- Detection of differentiation level of leukemic cells: Detection of surface markers of differentiation CD11b and CD14 in myeloid cell line HL-60 by means of flow cytometry and direct immunocytochemistry. Assessment of oxidative burst of monocytic cells stimulated by phagocytosis activators (zymosan, phorbol myristate acetate) by plate luminometer.
- Detection of differentiation of colon cancer cells (cell line HT-29): assessment of alkaline phosphatase by colorimetry (alkaline phosphatase cleaves colorless substrate 4-p-nitrophenyl phopshate producing yellow color).
- 2. Fluorescence and confocal microscopy:
- Principle and function of fluorescence microscope (advantages and limits), differences between conventional and confocal microscopy. Observation of 3D fixed interphase nuclei containing fluorescently labeled gene. Second fluorescent label stains the whole chromosome in which the target gene is localised.
- 3. Biochemical methods:
- Preparation of cell lysate for Western blotting and isolation of RNA:
Methods of cell lysate preparation - both for western blotting detection of proteins and isolation of RNA. Assessment of protein concentration using Bio-Rad DC kit. Electrophoresis and transfer of proteins to membrane - separation of proteins by SDS-PAGE, semi-dry electroblotting of proteins from gel to PVDF membrane
- Immunodetection of beta-actin by indirect immunochemistry (primary unlabelled anti-beta-actin Ab) and secondary antibody conjugated with horseradish peroxidase in samples from previous lecture. Visualisation of immunocomplex by ECL plus kit.
- Isolation of whole RNA, assessment of its concentration and quality:
Isolation of RNA from prepared cell lysate using the commercially available kit, assessment of concentration and purity of RNA using the Nanodrop ND-1000 Spectrophotometer device.
- ELISA (Enzyme Linked Immunosorbent Assay) technique:
Detection of TNF-alpha concentration in medium of differentiated THP-1 myeloid cells by commercially available Duoset kit from R&D system. Cells were treated for 24 hours by different concentrations of endotoxin.
- Cell culture and upstream processing. Edited by Michael Butler. 1st ed. New York: Taylor & Francis, 2007. xiv, 187 s. ISBN 0415399696. info
- Mammalian cell culture :essential techniques. Edited by A. Doyle - J. Bryan Griffiths. [1st ed.]. Chichester: John Wiley & Sons, 1997. 173 s. ISBN 0-471-97057-3. info
- Cancer cell culture :methods and protocols. Edited by Simon P. Langdon. Totowa, N.J.: Humana Press, 2004. xii, 360 s. ISBN 1-58829-079-4. 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
- Methods in cell biology :cell biological appplications of confocal microscopy. Edited by Brian Matsumoto. 2nd ed. Amsterdam: Academic Press, 2002. xii, 507 s. ISBN 0-12-580445-8. 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
- ELISA guidebook. Edited by John R. Crowther. 1st ed. Totowa: Humana Press, 2001. xi, 421 s. ISBN 0-89603-950-1. info
- Teaching methods
- Short theoretic lectures followed by practical course of selected specific methods; demonstrative introduction to advanced methodologies (high speed cell sorting, high resolution confocal microscopy, HPLC); homeworks; class discussion with skilled teacher.
- Assessment methods
- Individual evaluation of results and answering questions in protocols, final written test.
- Follow-Up Courses
- Further Comments
- The course is taught annually.
The course is taught: every week.
The course is also listed under the following terms
Autumn 2007 - for the purpose of the accreditation
Autumn 2010 - only for the accreditation