Individually Isolated Cells

The collected oocytes need to be individually isolated from the connective ovarian tissue and the layer of follicular cells that surround each oocyte (Goldin, 1992). For a detailed description, see Yao et al. (2000). Briefly, the excised ovarian lobe is washed in MBM and dissected using sterile forceps into smaller sections containing approximately 5-10 oocytes each. The sectioned clumps of oocytes are treated with MBM supplemented with 2 mg/mL type I collagenase (Worthington, Lakewood, NJ) for 2 h at room temperature with constant shaking to separate the oocytes from the follicular cell layer. The oocytes are subsequently washed five times with a BSA solution (bovine serum albumin, 1 mg/mL in MBM), followed immediately by five wash cycles in MBM. 

To investigate the individual compartments of the cell (see p. 196), various procedures have been developed to enrich and isolate cell organelles. These are mainly based on the size and density of the various organelles. 

FIGURE 12-35 Absorption spectra of purified rhodopsin and the red, green, and blue receptors of cone cells. The spectra, obtained from individual cone cells isolated from cadavers, peak at about 420, 530, and 560 nm, and the maximum absorption for rhodopsin is at about 500 nm. For reference, the visible spectrum for humans is about 380 to 750 nm. 

The immediate and reversible actions of dopamine are a combination of modulations of individual ion channels. The major ion channels expressed in spiny projection neurons are summarized in Table 2. Current understanding of the properties of these channels is mostly based on whole cell recordings from isolated cells, which have been recently reviewed by Nichola et al. (2000). The role of these channels in whole cell behavior has been studied using intracellular recordings in brain slices or anaesthetized animals. Many of the important cellular properties of spiny projection neurons can be accounted for in terms of ion channel activations occurring at different membrane potentials. 

While in vitro experiments offer a partial solution to the safety evaluation of chemicals, they cannot completely replace animal experiments for this purpose. Many chemicals are poisonous because they affect organs or systems as well as, or rather than, individual cells. The human body, like that of any mammal, is a highly complex, integrated system and reacts to chemicals in many different ways, far more than can be predicted from one or two different types of isolated cells. Most of the cells in our bodies have some similarities (for example, they all have a nucleus), but brain cells are very different from muscle cells, otherwise we would not be able to think Different types of cells have specialized functions which can make them a target for chemicals in specific ways. In particular, their metabolic capabihties are different and they may be unable to detoxify chemicals in the same way as hver cells, for example. This is illustrated by the story on p. 296. There is no doubt that the world would be a more hazardous place if the safety of chemicals were evaluated only in vitro. 

Bacteria that remained viable after 48 freeze-thaw cycles were used to initiate new cultures and these were subjected to additional freeze-thaw cycles as described. Individual isolates, which had been previously identified, as well as the control cultures, were also subjected to freeze-thaw cycles. Occasionally, single isolates in 10% TSB would supercool rather than freeze at temperatures close to 0°C, and to ensure that all samples froze at the same temperature, a few sterilized Agl crystals were added to the vials at the start of the experiments. For some experiments, cells were harvested by centrifugation (6,000 xg for 10 min) and the cell pellet washed with 10% TSB and kept at 0°C until analysis. Spent media was obtained by centrifuging, as above, and filtering (0.45 pm). 

Interpretation of the data is complicated by the presence of nonapoptotic cells with damaged membranes. Such cells may have phosphatidylserine exposed on plasma membrane and, therefore, similar to apoptotic cells, bind annexin V. Mechanical disaggregation of tissues to isolate individual cells extensive use of proteolytic enzymes to disrupt cell aggregates, remove adherent cells from cultures, or to isolate cells from tissue mechanical removal of the cells from tissue culture flasks (e.g., by a rubber policeman) and cell electroporation, may affect the binding of annexin V. Such treatments, therefore, may introduce experimental bias in subsequent analysis of apoptosis by this method. 

Fig. 2.4 Monoclonal antibodies. After injecting the antigen and generating several clones of antibodies, the spleen containing B-cells is removed. Hybridoma cells are made by fusing spleen B-ceUs with a myeloma cell culture line. To isolate the individual hybridoma cells producing one clone of antibody, the mixed hybridoma culture is highly diluted and plated in 96-well plates with one cell or less per well...

---