Fluorescent proteins expressed in mammalian cells can be quantified quickly and noninvasively with a standard fluorescence plate reader. We have previously exploited this quality in cell growth assessment (Hunt et al., 1999b). In this work, different CHO cell lines constitutively expressing fluorescent proteins were evaluated as model systems for process development and optimization. Our results demonstrate that the fluorescence of these cell lines quickly reveals conditions that might improve the overall productivity. Sodium butyrate, a well-known yet unpredictable enhancer of production, was chosen for this study. Due to the competing effects of sodium butyrate ("butyrate") on expression and cell number, the maximal overall productivity represents a compromise between enhancement of production and toxicity. Based on fluorescence only, it is possible to separate effects on cell number and specific production by combining microplate fluorescence measurements with data obtained by flow cytometry. This allows for rapid screening of different clones without counting cells or quantifying the recombinant protein, a highly attractive feature if the expression of green fluorescent protein (GFP) was correlated to that of a protein of interest. For all clones tested, negative effects of butyrate on proliferation were similar, while net enhancement of expression was characteristic for each clone. Therefore, it is necessary to optimize treatment for each individual clone. This work demonstrates that, based on the fluorescence of GFP-expresssing cell lines, it is possible to examine noninvasively three critical, generic parameters of butyrate treatment: butyrate concentration, exposure time, and culture phase at the time of addition.
Download full-text PDF |
Source |
---|---|
http://dx.doi.org/10.1002/bit.10108 | DOI Listing |
Biosens Bioelectron
January 2025
School of Chemistry and Molecular Engineering, East China Normal University, Shanghai, 200241, China. Electronic address:
The exploration of the mitochondrial apoptotic pathway in living cells is of great significance for achieving tumor diagnosis and treatment. However, visualization of the mitochondrial apoptotic pathway induced by specific proteins has rarely been reported. In this paper, we designed and synthesized a fluorescent probe Cy-JQ1 based on the bromodomain-containing protein 4 (BRD4) inhibition.
View Article and Find Full Text PDFSci Adv
January 2025
Center for Alzheimer's and Neurodegenerative Diseases, Peter O'Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Distinct tau amyloid assemblies underlie diverse tauopathies but defy rapid classification. Cell and animal experiments indicate tau functions as a prion, as different strains propagated in cells cause unique, transmissible neuropathology after inoculation. Strain amplification requires compatibility of the monomer and amyloid template.
View Article and Find Full Text PDFACS Appl Bio Mater
January 2025
Department of Chemistry, Indian Institute of Technology Palakkad, Palakkad, Kerala 678 623, India.
The aggregation of proteins, peptides and amino acids has been a keen subject of interest owing to their implications in metabolic disorders. In this work, we investigated the self-aggregation of the unmodified aromatic amino acid l-tryptophan (Trp) into unusual spherical microstructures. Using fluorescence spectroscopy and field emission scanning electron microscopy (FE-SEM), we detail the time-dependent transformation of monomeric tryptophan into spherical aggregates with distinct fluorescence characteristics (λ = 345 nm, λ = 430 nm) compared to the monomer.
View Article and Find Full Text PDFProc Natl Acad Sci U S A
January 2025
Department of Chemical and Biological Engineering, University of Colorado, Boulder, CO 80305.
Immunological interventions, like vaccinations, are enabled by the predictive control of humoral responses to novel antigens. While the development trajectories for many broadly neutralizing antibodies (bnAbs) have been measured, it is less established how human subtype-specific antibodies develop from their precursors. In this work, we evaluated the retrospective development trajectories for eight anti-SARS-CoV-2 Spike human antibodies (Abs).
View Article and Find Full Text PDFProtein Sci
February 2025
Laboratory MIVEGEC (Univ. Montpellier, CNRS, IRD), French National Center for Scientific Research (CNRS), Montpellier, France.
Biochemistry textbooks describe eukaryotic mRNAs as monocistronic. However, increasing evidence reveals the widespread presence and translation of upstream open reading frames preceding the "main" ORF. DNA and RNA viruses infecting eukaryotes often produce polycistronic mRNAs and viruses have evolved multiple ways of manipulating the host's translation machinery.
View Article and Find Full Text PDFEnter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!