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)
RNDr. Miroslav Machala, 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. Eva Bártová, Ph.D., DSc. (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.

Timetable

Fri 8:00–9:50 BFU

Prerequisites

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 13 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

Syllabus

• 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:
• 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.

Literature

• Cell culture and upstream processing. Online. Edited by M. Butler. 1st ed. New York: Taylor & Francis, 2007. xiv, 187. ISBN 9780415399692. [citováno 2024-04-23] info
• Mammalian cell culture : essential techniques. Online. Edited by A. Doyle - J. B. Griffiths. Chichester: John Wiley & Sons, 1997. xv, 173. ISBN 0471970573. [citováno 2024-04-23] info
• Cancer cell culture : methods and protocols. Online. Edited by Simon P. Langdon. Totowa, N.J.: Humana Press, 2004. xii, 360. ISBN 1588290794. [citováno 2024-04-23] info
• Animal cell culture :a practical approach. Online. Edited by R. Ian Freshney. 2nd ed. Oxford: Oxford University Press, 1992. xix, 329 s. ISBN 0-19-963213-8. [citováno 2024-04-23] info
• Cell biological appplications of confocal microscopy. Online. Edited by Brian Matsumoto. 2nd ed. Amsterdam: Academic Press, 2002. xii, 507. ISBN 0125804458. [citováno 2024-04-23] 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. Online. Totowa, N.J.: Humana Press, 2001. xi, 421. ISBN 0896037282. [citováno 2024-04-23] 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.

Language of instruction

Czech

Follow-Up Courses

• Bi5611c Special methods of animal physiology

Further Comments

Study Materials
The course is taught annually.

• Enrolment Statistics (Autumn 2011, recent)
  
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