Cell proliferation staining

Fig. 10.19 Lack of toxic effects of CM fullerene on breast epithelial cells. Cm does not inhibit cell proliferation. MCF 10A and (A) MDA MB 231 (B) breast cancer cell lines were cultured either in the absence or presence of methanol C60 and cell proliferation was assayed by crystal violet staining. Control, no C 1 Opg Cm, A 50 pg Cm, X 250 pg CM. (C). MDA MB 231 cells were simultaneously stained with calcein and ethidium using a live-dead assay kit. Lack of red-colored cells and the presence of cells stained in green indicate the lack of toxicity (D). MDA MB 231 cells were either untreated (open box) cultured with varying amounts 10 (gray ), 50 (patterned ) and lOOpg (filled ) of C60 for 48 h and analyzed for cell cycle progression by flow cytometry (Levi et al., 2006) (See Color Plates)...

Cell cycle dynamics are closely connected to cell growth and to the mechanism of controlling cell proliferation. The cell cycle can be defined as an ordered set of biochemical events resulting in cell division. The sequence of these events is divided into four phases the Gi phase, followed by the S phase (DNA synthesis), G2 phase and the M phase. For determining percentage of cells at different phases of the cell cycle, cells must be stained for DNA content with propidium iodide (PI) [20, 21], Based on the amount of DNA content by PI, the fraction of cells in a specific phase can be determined [22] from whole population using dedicate software (e.g, Modfit or Flowjo ) or, more precise, by using 2D-dot plot BrdU incorporation [23],... 

In order to confirm the increased postischemic cell proliferation with an independent marker, we stained monkey brain sections for Ki67 (Kee et al. 2002). Ki67 immunohistochemistry confirmed that proliferation was increased on day 9 (Fig. 9B) as compared to control (Fig. 9A), and subsequently declined (Fig. 9C). The Ki67+ cells were frequently grouped similarly to the BrdU+ cells, in clusters or doublets (Fig. 9D, E). Double-labeling for BrdU and Ki67 demonstrated they stain the same cells (Fig. 9F). BrdU also colabeled with the mitotic marker phosphohistone H3 (Fig. 9G). 

Fig.48A-C Neuronal progenitor and microglial cell proliferation in SVZa. A Double-staining for BrdU and / III-tubulin in postischemic day-23 ventral SVZa. The framed section is magnified in the lower panel. Most BrdU+ nuclei colabel with / III-tubulin+ chains of cells (arrows). B Double-staining for BrdU and /3111-tubulin in postischemic day-44 caudate SVZa. Some of the cells within a / III-tubulin+ cluster express BrdU (arrows). Note the perivascular position of the cluster (bv, blood vessel). C Double-staining for BrdU and Ibal in postischemic day-44 caudate SVZa. A BrdU+/Ibal+ cell (arrows) and a BrdU /Ibal+ cell (arrowheads) are depicted. Asterisk, anterior horn of lateral ventricle. Scale bars = 50 pm (A) 20 pm (B) 10 pm (C)...

The use of either MTT or MTS shows a linear relationship between cell number and the amount of formazan product produced. However, the ability to convert MTT or MTS to a formazan product may vary with cell type, and depends on a cell s metabolic capacity. Most eukaryotic cells yield significant level of formazan product from MTT or MTS at low cell numbers (Smail et al., 1992). The method for MTT staining is described below. It is however advisable and more practical to use the ready-to-use cell proliferation kits from Promega, which are based on either MTT or MTS staining. 

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