5-carboxytetramethylrhodamine-succinimidyl-ester and Colorectal-Neoplasms

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration in more appropriate 3D environments, which better resemble the dimensionality of the physiological processes in question. Migratory cells can substantially differ in their morphology and mode of migration depending on whether they are moving on 2D or 3D substrates. Due to technical difficulties and incompatibilities with most standard protocols, structural and functional analysis of cells embedded within 3D matrices still remains uncommon. This article describes methods for preparation and imaging of 3D cancer cell cultures, either as single cells or spheroids. As an appropriate ECM substrate for cancer cell migration, we use nonpepsinized rat tail collagen I polymerized at room-temperature and fluorescently labeled to facilitate visualization using standard confocal microscopes. This work also includes a protocol for 3D immunofluorescent labeling of endogenous cell cytoskeleton. Using these protocols we hope to contribute to a better description of the molecular composition, localization, and functions of cellular structures in 3D.

Topics: Animals; Cell Culture Techniques; Cell Movement; Collagen Type I; Colorectal Neoplasms; Cytoskeletal Proteins; Cytoskeleton; Fluorescent Antibody Technique; Fluorescent Dyes; Mice; Microscopy, Confocal; Neoplasm Proteins; Rats; Rhodamines; Spheroids, Cellular; Tumor Cells, Cultured

We developed a quantitative reverse-transcription polymerase chain reaction (RT-PCR) to determine CK20 expression in colorectal tumor and hematopoietic tissue.. Our method incorporates a calibrated PCR with an internal competitor and an external standard.. The RT-PCR assay is sensitive detecting 10 target molecules of CK20 in solution with one round of 38 amplification cycles. Genomic DNA contamination was eliminated by Dnase I digestion of total RNA. The inclusion of a calibrator in the quantitative RT-PCR analysis allowed for a high throughput of unknown samples within the same assay improving comparative analysis between the samples tested. Analysis of peripheral blood and bone marrow from 20 healthy volunteers revealed a low level of CK20 expression in all samples.. To study the clinical significance of CK20 expression as a marker of systemic metastatic disease it is essential to measure CK20 mRNA levels in hematopoietic tissue with sensitive quantitative RT-PCR. A sensitive and reproducible method, which is easily performed, is described.

Topics: Biomarkers, Tumor; Bone Marrow Cells; Calibration; Colorectal Neoplasms; DNA; Electrophoresis, Capillary; Fluorescent Dyes; Gene Expression; Humans; Intermediate Filament Proteins; Keratin-20; Reverse Transcriptase Polymerase Chain Reaction; Rhodamines; RNA, Messenger; Sensitivity and Specificity