BIOLOGY 3326F
Cell Biology Laboratory
Instructor: Dr. Alex Timoshenko
Course format: 3+ lab hours and 1 lecture/tutorial hour per week.
Prerequisite(s): Biochemistry 280a or the former Biology 280a; Biology 282b. Restricted to students with a minimum 70% in Biology 282b.
BIOLOGY 326F/G is a laboratory course which provides training in current cell biological methods such as a regular (up-right and inverted) and fluorescent microscopy, animal cell culture, cell adhesion, karyotype analysis, cytochemistry, fluorescent (immuno)staining. An important part of this course focuses on application of the mentioned methods to study cytoskeleton organization in BHK-21 fibroblast cells and to analyze cytoskeleton rearrangements in response to heat shock.
A public mailing list bio-326@uwo.ca will be used to send regularly e-mail messages (any updated information, instructions, and FAQs) to all students registered for the course.
Tutorial slides, lab results, and grades will be available through WebCT.
Recommended readings:
1. Freshney, R.I. Culture of Animal Cells: A Manual of Basic Technique, Fifth Edition. Willey-Liss, 2005.
2. Lodish, H., et al. (Eds.) Molecular Cell Biology, Sixth Edition. W.H. Freeman and Company, NY, 2008.
3. K. Knisely, A Student Handbook for Writing in Biology, Second Edition, W.H. Freeman and Company, 2005.
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Structure of the course:
The Bio326F/G course consists of two parts with the following training objectives:
Part I. Basics of Animal Cell Culture:
• Examination of the microscope potential (upright vs inverted; numerical aperture; magnification; resolution; field of view; working distance) •
Proper alignment and set-up of a bright-field light microscope (Köhler illumination) and a phase contrast microscope • Identification of cells and cellular components on slides and in cell culture flasks • Introduction to sterile techniques in cell culture • Harvesting methods for continuous cell lines growing in monolayer • Cell counts and estimation of cell density using hemocytometer • Test for cell viability • Monitoring morphology of animal cells in culture • Distinction between sterile and contaminated, and healthy and unhealthy cultures • Subculture of a continuous cell line, split ratio • Cell line characterization by growth kinetics • Plotting and analyzing a growth curve, doubling time • Cell-substrate adhesion • Preparation of chromosome spreads from monolayer cell cultures • Staining procedure for chromosome spreads with Giemsa • Karyotype analysis of a cell line • Modal number of chromosomes and heteroploidy • Aneuploidy • Genetic instability in cell culture.
Part II. Dynamics of the cytoskeleton:
• Staining of the cytoskeleton structures in monolayer cell cultures with Coomassie Brilliant Blue R250 (Pena’s method) • Fixation procedure and fixatives • Preparation of slides with stained cells • Fluorescence microscopy • Fluorochromes • Direct fluorescence staining of actin microfilaments with phalloidin • Preparation of slides for fluorescent microscopy • Monoclonal and polyclonal antibody • Primary and secondary antibody • Indirect immunofluorescence staining • Blocking solution • Observation of microfilament, microtubules and intermediate filament modifications in response to cytoskeletal drugs and hyperthermia and interpretation of these data.
