Cell-free System
Cell-free System - Related Videos
10:54
Quantitative Analysis of Cell Edge Dynamics during Cell Spreading
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Cited by 3 •
2021
In this protocol, we present the experimental procedures of a cell spreading assay that is based on live-cell microscopy. We provide an open-source computational tool for the unbiased segmentation of fluorescently labeled cells and quantitative analysis of lamellipodia dynamics during cell spreading.
07:20
Bioenergetic Profile Experiment using C2C12 Myoblast Cells
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Cited by 190 •
2010
A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.
09:43
Measurement of Heme Synthesis Levels in Mammalian Cells
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Cited by 12 •
2015
Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.
11:04
Control of Cell Geometry through Infrared Laser Assisted Micropatterning
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2021
The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.
12:16
Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma
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Cited by 22 •
2013
This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).
FAQs
DNA or RNA templates provide the information that the available molecular machinery can process. In gene-expression experiments, transcription components generate RNA from DNA, while translation machinery uses the resulting RNA to produce protein. Because these activities occur outside intact cells, researchers can examine or engineer gene-expression processes without the additional complexity of whole-cell growth.
Cell extracts or purified molecular components supply the machinery required for biological reactions, while energy sources and cofactors support that machinery. The choice between an extract and defined components affects how much of the reaction environment researchers can control. Together, these ingredients determine whether the system can carry out transcription, translation, or other selected cellular functions.
Removing intact cells separates the target biochemical activity from cellular growth constraints and other whole-cell processes. Researchers can therefore focus on a selected reaction and adjust its molecular inputs in a controlled environment. This isolation helps clarify mechanisms, supports testing of engineered pathways, and makes the resulting biological activity easier to connect with defined experimental conditions.
A typical setup combines a DNA or RNA template with cell extract or purified transcription and translation components, then supplies the required energy sources and cofactors. The assembled reaction is allowed to carry out the selected gene-expression process, after which researchers examine the RNA or protein produced. The exact mixture depends on whether transcription, translation, or both are being studied.
Researchers may choose this approach when they need rapid protein synthesis, want to test a metabolic pathway, or need to develop a biosensor or diagnostic without the growth constraints of whole-cell cultures. It is also useful when isolating a molecular mechanism or rapidly evaluating an engineered biological function is more important than maintaining living cells.
These systems can produce specific RNA or proteins and support testing of metabolic pathways, biosensors, diagnostics, and programmable biological technologies. The resulting activity provides information about whether selected molecular components can execute the intended function outside a living cell. In synthetic biology, that makes the platform useful for evaluating engineered cellular functions in a controlled setting.