Cell cycle analysis

Bryant As you showed on the growth curve for the imaginal disc, there is something that happens at the end of its development that stops both growth and proliferation. When you do your cell cycle analysis, is this in discs that have shut down at that point or in discs that are still in the growth phase ... 

For cell cycle analysis, HOS cells (1 x 106) were treated with the indicated concentrations (see Table 13.2) of MTX or MTX-LDH for 20h, harvested by trypsin treatment, washed with PBS, and then fixed with cold 70 % ethanol on ice overnight. The fixed cells were incubated with propidium iodide and flow cytometric measurement was carried out. Over 20 h, an increase in the number of cells in the Gl phase resulted from MTX and MTX-LDH treatment compared to untreated cells, indicating arrest at the Gl/S boundary. It is worth noting that the inhibition of the Gl/S transition was more evident in the cells treated with MTX-LDH than in those treated with free MTX (85.59% versus 66.62% at 320pM/ml). This is consistent with... 

Cell Cycle Analysis and Kinetics Apoptosis Calcium Flux Chromosome Analysis Micronuclei Analysis Intracellular pH Intracellular Glutathione Oxidative Burst Cell Viability... 

One of the first applications developed for flow cytometry was cell cycle analysis.2 There are numerous intercalating fluorescent DNA and RNA staining reagents that can be used to determine the amount of DNA in cells, an indicator of cell cycle stage and progression, as demonstrated in Figure 7.3. Nucleic acid dyes may be selective for DNA... 

Three C60 derivatives with two to four malonic acid groups (DMA C60, TMA C60, and QMA C60) were prepared and the phototoxicity of these compounds against HeLa cells was determined by MTT assay and cell cycle analysis (Yang et al., 2002). The relative phototoxicity of these compounds was DMA C60 > TMA C60 > QMA C60. Hydroxyl radical quencher mannitol (lOmM) was not able to prevent cells from the damage induced by irradiated DMA C60. DMA C60, together with irradiation, was found to decrease the number of G(l) cells from 63% to 42% and increase G(2) + M cells from 6% to 26%. 

Our previous studies showed that CNTs exhibit marked cytotoxicity (Cui et al., 2005 Tian et al., 2006). For example, SWCNTs can inhibit HEK293 cell proliferation, decrease cell adhesive ability in a dose-and time-dependent manner as shown in Fig. 9.7. HEK293 cells also exhibit active responses to SWCNTs such as secretion of some 20-30 kd proteins to wrap SWCNTs, aggregation of cells attached by SWCNTs and formation of nodular structures as shown in Fig. 9.8. Cell cycle analysis showed that 25pg/ml SWCNTs in medium induced Gt arrest and cell apoptosis in HEK293 cells as shown in Fig. 9.9. Biochip analysis showed that SWCNTs can induce up-regulation expression of cell cycle-associated genes such... 

Cell cycle analysis by dedicate software (e g. Modfit or Flowjo ) [26] usually underestimates percentage of cells in S-phase, since Gi and G2/M peaks are fitted by a gaussian model with modelling of cell cycle phases, and early (ES) and late S-phase (LS) are included inside fitted peaks (Figure 4). 

Cell-cycle analysis, combined with previous research, suggests that edotecarin may bind to topi with high affinity in cells that are not actively synthesizing DNA and may remain attached to topoisomerase I to induce cycle arrest and cell death once the S phase begins. 

Purdue University Cytometry Laboratories, West Lafayette, IN (February 14, 2005) http //www.cyto.purdue.edu/ Rabinovitch PS. 1994. DNA content histogram and cell-cycle analysis. Methods Cell Biol 41 263-296. 

Wersto RP, Chrest EJ, Leary JF, Morris C, Stetler-Stevenson MA, et al. 2001. Doublet discrimination in DNA cell-cycle analysis. Cytometry 46 296-306. 

Additionally, two studies have measured colorectal epithelial cell proliferation and apoptosis in human non-neoplastic mucosa in combination with serum bile acid quantification. Ochsenkuhn et al have reported a positive correlation between serum DCA levels and proliferation measured by flow cytometric cell cycle analysis. However, a more recent study of colorectal adenoma patients failed to detect a correlation between serum DCA and immuno-histochemical Ki-67 antigen labelling. Instead, this latter study revealed a positive correlation between serum DCA and the degree of TUNEL-positive epithelial cell apoptosis. ... 

N5. Noronha, A., and Richman, D. R, Simultaneous cell surface phenotype and cell cycle analysis of lymphocytes by flow cytometry. J. Histochem. Cytochem. 32, 821-829 (1984). 

Fig. 9. The cell cycle. M mitosis G1+S+G2 interphase, i.e., the period between ceU division. During G1 (G for gap) the cellular genome is in the diploid state. This is followed by the S-phase (S for synthesis) during which the DNA is replicated, and finally G2. Insert Cell-cycle analysis of HM02 cells cultured with and without vinblastine. CeU-cycle distribution was determined by staining DNA with propidium iodide, and the number of cells in different phases of the cell cycle was measured with a FACStar flow cytometer. Vinblastine, which disrupts the formation of microtubules, causes cell-cycle arrest in the G2/M-phase...

Schippers, K. J., Martens, D. E., Pomponi, S. A., and Wijffels, R. H. (2011). Cell cycle analysis of primary sponge cell cultures. In Vitro Cell. Dev. Biol. Anim. 47,302-311. 

Fig. 3. Cell cycle analysis of BrdU/DNA profiles. The curve is generated from narrow regions set in mid-S phase. One region is set around the BrdU-labeled cells and the other encompasses all cells in mid-S The y-axis is calculated from the ratio of cells in the BrdU-labeled compartment divided by that in the total mid-S region (R4 in the gated and ungated DNA profiles in Fig 2).

Pallavicini, M. G (1987) Solid tissue dispersal for cytokinetic analyses, m Techniques in Cell Cycle Analysis (Gray, J. W and Darzynewicz, Z., eds.), Humana, Clifton, NJ, pp. 139-162. 

Dolbeare, F, Kuo, W. L., Vanderlaan, M., and Gray, J W (1988) Cell cycle analysis by flow cytometric analysis of the incorporation of lododeoxyuridme (IdUrd) and bromodeoxyuridine (BrdUrd) Proc Am Assoc Cancer Res 29, 1896-1901... 

Fig. 5. Comparison of two-dimensional light scatter assay with subdiploid DNA peak assay. Burkitt lymphoma cells were serum-deprived for 14 d and then assayed for (A) forward light scatter (FSC) versus 90° light scatter (SSC) or (B) cell cycle. Note that although all the cells are clearly apoptotic as shown by the light scatter assay, there is only a small subdiploid peak (SD) present in the cell-cycle analysis, demonstrating that in this case, celt-cycle analysis alone would grossly underestimate the extent of apoptosis present in the cell population.

The nick translation method can be used in conjunction with cell-cycle analysis to determine which phases of the cell-cycle the apoptotic cells are in. To do this, add 5 pg/mL propidium iodide to the samples 30 min prior to analysis. 

Gerdes, J., Lemke, H, Baisch, H, Wacker, H -H, Schwab, U, and Stem, H. (1984) Cell-cycle analysis of a proliferation-associated human nuclear antigen defined by the MAb Ki-67. J Immunol 133, 1710-1715... 

We and others have demonstrated facile synthesis of a number of new anticancer active p-lactams. The p-lactam derivatives described herein are unique, and they demonstrate reasonable in vitro antitumor cytotoxicity. The stereochemical outcome of the Staudinger reaction as reported herein may offer our laboratory and others many additional opportunities to use p-lactams in the synthesis of biologically active compounds. Although the mechanism of action of the lead compounds has not been totally established, our research on cell cycle analysis offers intriguing... 

Single nucleotide polymorphisms (SNPs), 218 Software, 43-46 for aggregate subtraction, 137 for cell cycle analysis, 135-137 for spectral compensation, 79 Soluble analytes, 218-220 Sorting, 159-173 accuracy of, 166, 168 alternate methods for, 170-172 batch, 170-171 break-off point, 161-163 efficiency of, 168-170, 169f for prenatal diagnosis, 221 high speed, 165-166, 169-170, 172, 215, 225... 

Dual staining of cells can also be used to look at DNA and protein markers simultaneously. Using methods similar to those described in the previous chapter for looking at cytoplasmic proteins simultaneously with surface membrane proteins, cells can be stained for surface proteins, then fixed and permeabilized, and then stained for DNA. Figure 8.16 shows the way that this type of technique can be used to permit the cell cycle analysis of subpopulations of cells independently. In this particular example, it can be seen that, after treatment with the mitogen phytohemagglutinin, it is the CD8-positive cells more than the CD8-negative cells that have been induced to enter S phase. 

Fig. 8.16. Cell cycle analysis of CD8-positive and CD8-negative cells. Lymphocytes were cultured with phytohemagglutinin, stained with FITC anti-CD8 monoclonal antibody, treated with saponin to permeabilize the outer membrane, and then stained with propidium iodide (PI) and RNase. Cells provided by Ian Brotherick.

Several chapters in Ormerod and several sections in the Purdue Handbook, in Diamond and DeMaggio, and in Current Protocols in Cytometry give good practical protocols. Darzynkiewicz (both the 1994 and 2001 editions) include many chapters with protocols and advice on both the simpler and the more complex methods in nucleic acid, apoptosis, and cell cycle analysis. 

Good discussions of the mathematical algorithms for cell cycle analysis can be found in Chapter 6 of Van Dilla et al., Chapter 23 of Melamed et al., and in the multiauthor book Techniques in Cell Cycle Analysis, edited by Gray JW and Darzynkiewicz Z (1987), Humana Press, Clifton, NJ. 

Cell Cycle Analysis 131 Two-Color Analysis for DNA and Another Parameter 142 Chromosomes 147 Apoptosis 150 Necrosis 154... 

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