cell growth, differentiation and senescence

[...]... that protrude from the cell surface, such as cellular receptors A 10-15 min incubation at 37°C is required to cause cell detachment and disrupt cell- cell adhesion.] 6 Triturate the detached cells with a 2 ml pipette several times to disrupt cell clumps and effect a single cell suspension 7 Add 4 ml medium containing 10% fetal calf serum to inactivate the trypsin and triturate the cells several times to... outlines the steps for counting cells in tissue culture The measurement of cell number should take place in the presence of a dye, such as trypan blue, excluded only by viable cells By counting both blue and white cells, an estimate of cell viability can also be obtained Percent viability = white cells/( blue cells + white cells) X 100% Protocol 1 Counting cell numbers Equipment and reagents • Ca2+, Mg2+-free... 6 Summary 278 References 279 14 Senescence and immortalization of human Cells 281 Karen Hubbard and Harvey L Ozer 1 Introduction 281 2 Cellular replicative senescence 282 3 SV40 transformation and crisis 287 4 SV40-immortalized cell lines 290 5 Telomeres and telomerase 293 6 Conditional SV40 transformants 296 xv Contents 7 Other approaches to immortalization of human cells 297 8 Summary 298 References... of the cells undergoing cell cycle progression minus the population of cells simultaneously undergoing cell death It can also be reflected in the length of time it takes cells to traverse Table 1 Assays for parameters of proliferation Property measured Assay 1 Cell number a Rate of division exclusive of cell death b Percent viable cells c Doubling time a Fraction of cells in specific phases of cell cycle... the fraction of cells in specific phases of the cell cycle and cells that have undergone cell death and DNA fragmentation and loss After various interventions, cells are harvested by trypsinization or from suspension cultures, labelled with propidium iodide (PI) or another DNA-binding dye, as in Protocol 3, and assayed by flow cytometry The technical aspects of using a flow cytometer and programmes... procedure, mitotic cells have a much lower scatter than G2 phase cells, while Gl post-mitotic cells have lower scatter than Gl cells ready to enter the S phase The procedure is outlined in Protocol 4 Protocol 4 Flow cytometric differentiation between G2 and M and between post-mitotic and pre-synthetic G1 (from ref 5) Equipment and reagents • • • BrdU PBS RNase A 0.1 M HCI PBST: 5 ml ice-cold PBS and 0.4% Tween-20... of ONs into cells 5 Conclusions References 217 220 220 222 223 11 Human leukaemia cells as a model differentiation system 225 Dorothy C Moore and George P Studzinski 1 Introduction 2 Cell types 3 Assessment of monocytic differentiation 4 Assessment of erythroid differentiation 5 Assessment of megakaryocytic differentiation 6 Summary References 225 225 226 230 236  239 239 12 Induction of differentiation. .. often off by a small factor in benign cells owing to the presence of a small fraction of cells in the S and G2+M phases, but is even more inaccurate in cells from tumour tissue where more cells are cycling and a significant fraction of the cells may also be aneuploid The method of extracting DNA and measuring its content is outlined in Protocol 2 Protocol 2 Extraction and measurement of DNA (from ref 2)... of cells dividing cells Cell cycle distribution and Flow cytometry 2 DNA content fraction in S phase 3 Ki67, PCNA, AgNOIR Related to fraction Immunohistochemistry of cells proliferating 2 DNA synthesis a IHC = immunohistochemistry 3 Robert Wieder various phases of the cell cycle and the effects of various interventions on each phase Various aspects of the cell cycle are affected by interventions, and. .. ug/ml) in PBS and acquire the samples in the flow cytometer Figure 1b demonstrates the cytofluorometric distribution of cells by their DNA content The cell fit programme (CELLQuest Version 2.0 program, Becton Dickinson Immunocytometry Systems, San Jose, CA) calculated the fraction of cells with 2n, 2n to 4n, and 4n amounts of DNA, corresponding to G1, S, and G2+M phases of the cell cycle While cell counting . Analysis (2nd edition) Cell- Cell Interactions The Cell Cycle Cell Growth and Apoptosis if Cell Separation Cellular Calcium Cellular Interactions in Development Cellular Neurobiology * . class="bi x0 y0 w0 h1" alt="" Cell Growth, Differentiation and Senescence The Practical Approach Series SERIES EDITOR B. D. HAMES Department of Biochemistry and Molecular Biology University . Hubbard and Harvey L. Ozer 1. Introduction 281 2. Cellular replicative senescence 282 3. SV40 transformation and crisis 287 4. SV40-immortalized cell lines 290 5. Telomeres and