Flow cytometry experiments

The following basic protocols may be used alone or combined with other staining procedures in multiparameter flow cytometry experiments. Although they are illustrated with data from cells that proliferate in suspension, these protocols may be easily modified for the analysis of cells isolated from tissues or adherent cells in culture, by incorporating an initial step for the preparation of single cell suspensions. The assays are conducted at room temperature, unless otherwise noted. 

For FACS experiments, DNA concentrations between 1 and 10 jig/mL are suggested with peptide to DNA ratios between 0 and 10 (w/w). For microscopy experiments, we recommend using higher DNA concentrations (5-20 p,g/mL) and similar peptide to DNA ratios as those used in the flow cytometry experiments. 

For passaging and seeding, the cells are first washed with PBS and detached from the culture flask by incubating with 3 mL trypsin/EDTA solution for approximately 10 min in the incubator. The trypsin/EDTA solution is inactivated by adding 13 mL of complete culture medium. For maintenance, 1 10 part is transferred into a new culture flask. For seeding or for use in a flow cytometry experiment, the cells are centrifuged (5 min, 300 x ), resuspended in the required medium, counted and diluted to the appropriate number of cells/ml. 

To sort a library, include at least tenfold more yeast in your sample than the theoretical size of your library (if possible). For example, if your library size is 107 unique yeast, make sure you induce and stain at least 108 yeast. To perform an analytical flow cytometry experiment, smaller samples may be used, around 106 yeast per staining. 

The result of a flow cytometry experiment is a histogram of cell distribution by the total amount of surface-bound chemokine, with or without an unlabeled competitor Hgand. This assay provides an easy way to quickly screen numerous candidate disulfide-trapped construct combinations uHth-out the complexities of sample purification however, the utUity of this method is limited to chemokines that are detectable on the cell surface by a... 

There is a single assumption in these measurements--namely that the antibody only quenches free ligand. This has been demonstrated specifically by flow cytometry in experiments which show that there is no quenching of ligand on the cell (3). The kinetic analysis depends on rapid interaction of ligand and antibody, which in these experiments is essentially within the mixing time. 

Fig. 11.7 Effect of HU on ml-CAM-1 expression in the TrHBMEC (a) and EA-hy 926 (b). These cells were incubated with HU 250 pM for 48 h with or without 100 U mb of TNFaand IFNy. mlCAM-1 cellular expression was analyzed by flow cytometry. Results are the Mean Fluorescent Index (MFI) of one representative experiment, with overall trend in three other independent experiments being comparable. Parallel estimation of slCAM-1 release in the culture supernatant of TrHBMEC cells (6 independent experiments) revealed that without cytokines sICAM-1 was not detectable in the supernatant for the basal conditions. The results of HU-treated cells (c) in the presence of cytokines showed a significant increase in release of slCAM-1 (p <0.05).

To be used as delivery carriers for biomolecules, first it is essential to check whether the conjugates can effectively enter cells. Cellular uptake experiments were performed by using fluorescein isothiocyanate (FITC) conjugated LDH as a probe. Cells (5 x 105/ 1 ml) were incubated with LDH-FITC and its uptake was measured by flow cytometry. As shown in Figure 13.4, the cellular uptake was time and concentration dependent and... 

The above examples show that a very important criterion in the choice of a probe is its sensitivity to a particular property of the microenvironment in which it is located (e.g. polarity, acidity, etc.). On the other hand, insensitivity to the chemical nature of the environment is preferable in some cases (e.g. in fluorescence polarization or energy transfer experiments). Environment-insensitive probes are also better suited to fluorescence microscopy and flow cytometry. 

The experiment in Figure 13-3 illustrates the use of flow cytometry to determine Kj and B ax values for binding of the es nucleoside transporter ligand, SAENTA-fiuorescein, at sites in human CEM leukemic lymphoblasts. The es nucleoside transporter is widely expressed in mammalian cells, and is concentrated in regions in the CNS that coordinately express adenosine Ai receptors (Jennings et al., 2001). 

Furthermore, cell cycle analysis by flow cytometry can be improved by using other complementary techniques that provide additional information. This is the case of the mitotic index that informs us about the number of cells undergoing mitosis during the experiment. This measurement was also recorded in our investigation, and the results indicated its excellent complementary information. 

Apoptosis was measured by TdT-mediated dUTP nick end labeling (TUNEL) by flow cytometry as described by Gorczyca et al. (G6). Every measurement was done in duplo, and a negative and postive control were carried out with each experiment. All patients had normal basal FSH levels, and their partners had normal semen... 

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