Data analysis cell preparation

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. 

FIGURE 4 Densitometric analysis of Northern blots showing the effects of nitric oxide (NO) on the expression of monocyte chemoattractant protein (MCP-1) mRNA. Cultured human umbilical vein endothelial cells were incubated for 12 hr with solvent (control), the NO synthase inhibitor N°-nitro-L-arginine (L-NAG), or the NO donor SIN-1. Data were obtained using five different cell preparations. P < 0.01. 

In this chapter, we described an easily implemented, effective, and highly reproducible dual-column HPLC prefractionation method that we have developed for improving routine proteomic analyses. The approach results in multifold increases in the numbers of proteins that can be confidently identified by LC-MS of whole cell lysates without fractionation. We outline the key steps in the overall procedure, from sample preparation through to MS/MS and attendant data analysis, using yeast soluble protein extract as a test mixture. 

Refer to section Cell Setup and Connections for Three- and Two-Electrode Configurations for a three-electrode cell setup. The total preparation time is approximately 1 h and the experiment time requires anywhere from a few minutes to a few hours, depending on the number of potentials and frequencies used in the evaluation. The data analysis can be simple and straightforward for ideal samples, taking only a few minutes. Non-ideal M-S plots, however, can take much longer to analyze, and may require the researcher to move on to another technique to analyze E, . 

CD, JV, GY developed the cell culture and tissue sample preparation methods. SA, ST, and JM provided the Sd-FFF separation methods. All authors participated in the data analysis and manuscript preparation. 

This chapter describes practical aspects of the application of UV absorbance temperature profiles to determine the thermodynamics of nucleic acid structural transitions. Protocols and practical advice are presented for issues not normally addressed in the primary literature but that are crucial for the determination of reliable thermodynamics, such as sequence design, sample preparation, choice of buffer, protocols for determining strand concentrations and mixing strands, design of microvolume cuvettes and cell holder, instrumental requirements, data analysis methods, and sources of error. References to the primaiy literature and reviews are also provided where appropriate. Sections of this chapter have been adapted from previous reviews and are reprinted with permission from the Annual Review of Biochemistry, Volume 62 1993, by Annual Reviews wwwAnnualReviews.org (6) and with permission from Biopolymers 1997, by John Wiley Sons, Inc. (4). 

Bottom-up proteomics methods still need refinement of protocols, and improvements in the standardization and availability of bioinformatics tools for comprehensive data analysis on a routine basis. Although recent innovations in mass spec-trometric instramentation have aeeelerated the speed and sensitivity of proteome analysis (Hebert et al. 2014), further improvements can be obtained by emphasizing the optimization, simplification, and automation of sample preparation, for example, through single-tube proteomics approaches integrating all steps from cell lysis to peptide fractionation (Hughes et al. 2014 Fan et al. 2014), peptide separation techniques, and bioinformatics tools for fast, automated data interpretation for strain-level identification of cultivable bacteria and comprehensive characterization of each isolated microbial strain in the near future. 

---