Cell lysis assays

A prerequisite for CTL-mediated immune response is the formation of the MHC-class I-peptide complex and subsequent recognition by the T-cell repertoire, which can be analyzed in cell lysis assays with MCr-loaded target cells. Chromium release is a measure for peptide-induced cell lysis by CTL, and indicates the potency of the peptide to serve as an allele-specific epitope. A synthetic epitope has been identified with the peptide library approach to elucidate the molecular basis for the observed cross-recognition of two ligands by a single receptor [53]. 

Fig. 2 Biocompatibility assays of polyacetal 16 described in Scheme 5. (a) Cytotoxicity assay (B16F10 cells stimulated for 72 h) positive control was poly-L-lysine, negative control was Dextran. (b) Red blood cell lysis assay after 24 h positive control was poly(ethylene imine) (PEI), negative control was Dextran. Adapted with permission from [53], Copyright 2002 American Chemical Society...

The toxicity of TSST-1 and HI 35 A was tested in 20 g BALB/c mice (n=15 per dose) injected intraperitoneally (ip) with either protein diluted in PBS. Four hours later, mice were injected ip with 75 pg of E. coli 055 B5 LPS (Difco Laboratories, MI) and lethality was observed over 72 h. The serum cytokine levels of tumor necrosis factor a and P (TNF), as well as interferon gamma (IFNy), were measured over 14 h (2 h intervals) in mice injected with either 15 pg TSST-1, 15 pg TSST-1 plus 75 pgLPS, 15 pgH135Aplus LPS, or 75 pg LPS only. Serum levels of TNF were determined by an ELISA (Genzyme, M A) with triplicate samples the standard deviation (SD). Data for IFNy were collected from an L929 cell lysis assay with duplicate samples 10%. 

Determine the protein concentration of the soluble protein extracts by Bradford assay (20) to confirm that effective cell lysis has occurred see Note 5). 

The serum should be yellowish. A reddish color is indicative of erythrocytes lysis (hemolysis) which may interfere with clinical chemistry assays based on colorimetric values. If this happens, you may need to adjust the speed at which the blood is collected and processed or other steps that may cause sheer and red blood cell lysis. 

Schilling, E.A., Kamholz, A.E., Yager, P., Cell lysis and protein extraction in a microfluidic device with detection by a fluorogenic enzyme assay. Anal. Chem. 2002, 74(8), 1798-1804. 

Enzyme regulation and interpretation of activity assays A major uncertainty in using esterases to estimate cell lysis is the very broad nature of the assay. It is quite clear that FDA can be hydrolyzed by a number of different enzymes, including some of the extracellular enzymes describe above (e.g., Schnurer and Rosswall, 1982) obviously this is not compatible with the assumption that they are solely intracellular. Within cells, esterases are typically regulated by compartmentalization rather than other mechanisms, and so regulatory features are not likely to complicate interpretations of activity assays. 

Information gained from assays and future potential The application of esterases assays have provided the first direct evidence that cell lysis (as distinguished from loss terms such as grazing and sedimentation) may be an important factor in marine systems (see Kirchman, 1999). While the meaning of cell lysis, its incidence and ecological importance remain unclear (see Franklin et al, 2006), and there are concerns about the assay, we currently lack objective, independent methods with which to measure lysis (cf, Agusti and Duarte, 2002). In future, it may be possible to identify particular esterases that are truly found only within cells and choose substrates that are more specific for them. 

The most commonly used approaches are the neutral red assay (cell viability and membrane damage), the Lowry (labeled proline), Coomassie blue, and Kenacid blue assays (cell proliferation and total cell protein), the MTT or tetrazolium assay (mitochondrial function), and the intracellular lactate dehydrogenase activity test (cell lysis). 

Luciferase assay Cell lysis buffer (0.1 M Tris-HCl (pH 7.8),... 

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