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Fluorescent derivatization of a protease antigen to track antigen uptake and processing in human cell lines.

ABSTRACT: BACKGROUND: We have devised a simple and efficient fluorescence-based method to track antigen uptake and processing in human B lymphoblastoid cells (B-LCL). Fluorescein labelled subtilisin was used to optimize antigen uptake conditions and identify processed peptides from human cell lines. RESULTS: Fluorescein labelled subtilisin conjugates had 0.06 to 2 moles of fluorescein per subtilisin molecule. High performance liquid chromatography and mass spectrometry (NanoESI-LC/MS/MS) analysis identified fluorescein conjugated to K141, K256, and the N terminus. Conjugates retained antigenic specificity to subtilisin specific antibodies and could be processed by whole cell extracts into low molecular weight fragments at pH 5.2. Maximal antigen uptake and processing occurred when PMSF (phenylmethylsulfonyl fluoride) inhibited subtilisin conjugate was incubated with cells at 100-200 microg/ml for 16 to 24 hr. Once optimal uptake conditions were established, processed subtilisin peptides were isolated and identified from human cell lines. CONCLUSION: Our studies show that FITC-conjugation provides an efficient tool to track the uptake and processing of this protease antigen and to facilitate identification of processed antigenic peptides from human cell lines.

SUBMITTER: Patil NS

PROVIDER: S-EPMC459215 | biostudies-literature | 2004 Jun

REPOSITORIES: biostudies-literature

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Fluorescent derivatization of a protease antigen to track antigen uptake and processing in human cell lines.

Patil Namrata S NS Wong David L DL Collier Katherine D KD McDonald Hugh C HC

BMC immunology 20040628

<h4>Background</h4>We have devised a simple and efficient fluorescence-based method to track antigen uptake and processing in human B lymphoblastoid cells (B-LCL). Fluorescein labelled subtilisin was used to optimize antigen uptake conditions and identify processed peptides from human cell lines.<h4>Results</h4>Fluorescein labelled subtilisin conjugates had 0.06 to 2 moles of fluorescein per subtilisin molecule. High performance liquid chromatography and mass spectrometry (NanoESI-LC/MS/MS) anal ...[more]

PMID: 15222895

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Project description:BackgroundThe lack of sensitive biocompatible particle track detectors has so far limited parallel detection of physical energy deposition and biological response. Fluorescent nuclear track detectors (FNTDs) based on Al?O?:C,Mg single crystals combined with confocal laser scanning microscopy (CLSM) provide 3D information on ion tracks with a resolution limited by light diffraction. Here we report the development of next generation cell-fluorescent ion track hybrid detectors (Cell-Fit-HD).MethodsThe biocompatibility of FNTDs was tested using six different cell lines, i.e. human non-small cell lung carcinoma (A549), glioblastoma (U87), androgen independent prostate cancer (PC3), epidermoid cancer (A431) and murine (VmDk) glioma SMA-560. To evaluate cell adherence, viability and conformal coverage of the crystals different seeding densities and alternative coating with extracellular matrix (fibronectin) was tested. Carbon irradiation was performed in Bragg peak (initial 270.55 MeV u?¹). A series of cell compartment specific fluorescence stains including nuclear (HOECHST), membrane (Glut-1), cytoplasm (Calcein AM, CM-DiI) were tested on Cell-Fit-HDs and a single CLSM was employed to co-detect the physical (crystal) as well as the biological (cell layer) information.ResultsThe FNTD provides a biocompatible surface. Among the cells tested, A549 cells formed the most uniform, viable, tightly packed epithelial like monolayer. The ion track information was not compromised in Cell-Fit-HD as compared to the FNTD alone. Neither cell coating and culturing, nor additional staining procedures affected the properties of the FNTD surface to detect ion tracks. Standard immunofluorescence and live staining procedures could be employed to co-register cell biology and ion track information.ConclusionsThe Cell-Fit-Hybrid Detector system is a promising platform for a multitude of studies linking biological response to energy deposition at high level of optical microscopy resolution.

Dataset Information

Fluorescent derivatization of a protease antigen to track antigen uptake and processing in human cell lines.

Publications

Fluorescent derivatization of a protease antigen to track antigen uptake and processing in human cell lines.

Similar Datasets

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