Abstract:
:Limbal stem cells (LSCs) have an important role in the maintenance of the corneal surface epithelium, and autologous cultured limbal epithelial cell transplantations have contributed substantially to the treatment of the visually disabling condition known as LSC deficiency. In this protocol, we describe a method of establishing human limbal epithelial cell cultures by a feeder-free explant culture technique using a small limbal biopsy specimen and human amniotic membrane (hAM) as the culture substrate. This protocol is free of animal-derived products and involves the use of human recombinant growth factors. In addition, the recombinant cell dissociation enzyme TrypLE is used to replace trypsin and autologous serum replaces FBS. It takes approximately 2 weeks to establish a confluent monolayer from which approximately 3 x 10(6) cells can be harvested. This procedure can be adopted for both basic research purposes and clinical applications.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Mariappan I,Maddileti S,Savy S,Tiwari S,Gaddipati S,Fatima A,Sangwan VS,Balasubramanian D,Vemuganti GKdoi
10.1038/nprot.2010.115subject
Has Abstractpub_date
2010-08-01 00:00:00pages
1470-9issue
8eissn
1754-2189issn
1750-2799pii
nprot.2010.115journal_volume
5pub_type
杂志文章相关文献
Nature Protocols文献大全abstract::Protein ubiquitination is a versatile protein modification that regulates virtually all cellular processes. This versatility originates from polyubiquitin chains, which can be linked in eight distinct ways. The combinatorial complexity of eight linkage types in homotypic (one chain type per polymer) and heterotypic (m...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2015.018
更新日期:2015-02-01 00:00:00
abstract::Detailed biochemical analysis of unmanipulated germinal center (GC) B cells has not been achieved. Previously, we designed and used a simple, economical and new magnetic bead separation scheme for the purification of 'untouched' mature GC and non-GC B cells from the spleens of immunized mice and reported the first bio...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2011.344
更新日期:2011-06-09 00:00:00
abstract::The combination of ChIP-seq and transcriptome analysis is a compelling approach to unravel the regulation of gene expression. Several recently published methods combine transcription factor (TF) binding and gene expression for target prediction, but few of them provide an efficient software package for the community. ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2013.150
更新日期:2013-12-01 00:00:00
abstract::The small ubiquitin-like modifier (SUMO) is a protein modifier that is post-translationally coupled to thousands of lysines in more than a thousand proteins. An understanding of which lysines are modified by SUMO is critical in unraveling its function as a master regulator of all nuclear processes, as well as its invo...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2016.082
更新日期:2016-09-01 00:00:00
abstract::Multiplex-fluorescence in situ hybridization (M-FISH) was initially developed to stain human chromosomes--the 22 autosomes and X and Y sex chromosomes--with uniquely distinctive colors to facilitate karyotyping. The characteristic spectral signatures of all different combinations of fluorochromes are determined by mul...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.160
更新日期:2006-01-01 00:00:00
abstract::Rapid evaluation of microbial cell-surface carbohydrates is essential to understanding the mechanisms by which bacteria use glycans to establish pathogenic or symbiotic relationships. Microbial glycan analysis is complicated both by the vast diversity of possible carbohydrate structures and by their dynamic nature. Ba...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.76
更新日期:2006-01-01 00:00:00
abstract::The colorimetric bio-barcode assay is a red-to-blue color change-based protein detection method with ultrahigh sensitivity. This assay is based on both the bio-barcode amplification method that allows for detecting miniscule amount of targets with attomolar sensitivity and gold nanoparticle-based colorimetric DNA dete...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2007.201
更新日期:2007-01-01 00:00:00
abstract::Apoptosis is a critical factor in AIDS and other viral illnesses, cerebral and myocardial ischemia, autoimmune and neurodegenerative states, organ and bone marrow transplant rejection, and tumor response to chemotherapy and radiation. Improved methods to identify sites of apoptosis are increasing our understanding of ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.17
更新日期:2006-01-01 00:00:00
abstract::This protocol describes the efficient, generally applicable Ullmann coupling reaction of bromaminic acid with alkyl- or aryl-amines in phosphate buffer under microwave irradiation using elemental copper as a catalyst. The reaction leads to a number of biologically active compounds. As a prototypical example, the synth...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2010.63
更新日期:2010-05-01 00:00:00
abstract::Pseudo-afferent cervical lymph-duct cannulation in a sheep model allows large amounts of lymph cells to be collected under physiological conditions, carrying immune signaling information from the head tissues, including oro-nasal mucosae. Importantly, large quantities of dendritic cells (DCs) of several subtypes are o...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.147
更新日期:2006-01-01 00:00:00
abstract::Unlike transplantation of other solid organs, vascularized mouse lung transplantation has only recently been developed. In this protocol, we describe a detailed method for performing a vascularized and aerated mouse orthotopic lung transplant, which to date represents the most physiological mouse model of lung transpl...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2008.218
更新日期:2009-01-01 00:00:00
abstract::Here we describe a protocol for the production of frozen competent yeast cells that can be transformed with high efficiency using the lithium acetate/single-stranded carrier DNA/PEG method. This protocol allows the production of highly competent yeast cells that can be frozen and used at a later date and is especially...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2007.17
更新日期:2007-01-01 00:00:00
abstract::Determining enantiomeric excess (e.e.) in chiral compounds is key to development of chiral catalyst auxiliaries and chiral drugs. Here we describe a sensitive and robust fluorescence-based assay for determining e.e. in mixtures of enantiomers of 1,2- and 1,3-diols, chiral amines, amino alcohols, and amino-acid esters....
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-020-0329-1
更新日期:2020-07-01 00:00:00
abstract::PatternLab for proteomics is an integrated computational environment that unifies several previously published modules for the analysis of shotgun proteomic data. The contained modules allow for formatting of sequence databases, peptide spectrum matching, statistical filtering and data organization, extracting quantit...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2015.133
更新日期:2016-01-01 00:00:00
abstract::The Golgi apparatus undergoes extensive disassembly during mitosis and reassembly in post-mitotic daughter cells. This process has been mimicked in vitro by treating Golgi membranes with mitotic and interphase cytosol. To determine the minimal machinery that controls the morphological change, we have developed a defin...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2010.38
更新日期:2010-04-01 00:00:00
abstract::This protocol describes a method for the extraction of DNA from elephant ivory. These techniques are being used to assign geographic origin to poached ivory by comparing the ivory genotype to a geographic-based gene frequency map, developed separately. The method has three components: ivory pulverization, decalcificat...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2007.318
更新日期:2007-01-01 00:00:00
abstract::The rodent malaria parasite Plasmodium chabaudi chabaudi shares many features with human malaria species, including P. falciparum, and is the in vivo model of choice for many aspects of malaria research in the mammalian host, from sequestration of parasitized erythrocytes, to antigenic variation and host immunity and ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2011.313
更新日期:2011-04-01 00:00:00
abstract::Mouse embryonic stem cells (mESCs) are key tools for genetic engineering, development of stem cell-based therapies and basic research on pluripotency and early lineage commitment. However, successful derivation of germline-competent embryonic stem cell lines has, until recently, been limited to a small number of inbre...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2014.030
更新日期:2014-03-01 00:00:00
abstract::High-throughput ballistic injection nanorheology is a method for the quantitative study of cell mechanics. Cell mechanics are measured by ballistic injection of submicron particles into the cytoplasm of living cells and tracking the spontaneous displacement of the particles at high spatial resolution. The trajectories...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2011.436
更新日期:2012-01-05 00:00:00
abstract::AMPylation (adenylylation) has been recognized as an important post-translational modification that is used by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes, and it is widely conserved across both prokaryotes and e...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2015.044
更新日期:2015-05-01 00:00:00
abstract::Unlike humans, mouse bone marrow-derived mesenchymal stem cells (MSCs) cannot be easily harvested by adherence to plastic owing to the contamination of cultures by hematopoietic cells. The design of the protocol described here is based on the phenomenon that compact bones abound in MSCs and hematopoietic cells exist i...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2009.238
更新日期:2010-03-01 00:00:00
abstract::In the past few decades, marine natural products bioprospecting has yielded a considerable number of drug candidates. Two marine natural products have recently been admitted as new drugs: Prialt (also known as ziconotide) as a potent analgesic for severe chronic pain and Yondelis (known also as trabectedin or E-743) a...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2008.182
更新日期:2008-01-01 00:00:00
abstract::Blood circulating endothelial cells (CECs) and circulating hematopoietic progenitor cells (CPCs) represent two cell populations that are thought to play important roles in tissue vascularization. CECs and CPCs are currently studied as surrogate markers in patients for more than a dozen pathologies, including heart dis...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2007.111
更新日期:2007-01-01 00:00:00
abstract::Human embryonic stem (hES) cells are self-renewing, pluripotent cells that are valuable research tools and hold promise for use in regenerative medicine. Most hES cell lines are derived from cryopreserved human embryos that were created during in vitro fertilization (IVF) and are in excess of clinical need. Embryos th...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2008.60
更新日期:2008-01-01 00:00:00
abstract::A big challenge in proteomics is the identification of cell-type-specific proteomes in vivo. This protocol describes how to label, purify and identify cell-type-specific proteomes in living mice. To make this possible, we created a Cre-recombinase-inducible mouse line expressing a mutant methionyl-tRNA synthetase (L27...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-018-0106-6
更新日期:2019-02-01 00:00:00
abstract::This protocol describes pulsed-field gel electrophoresis (PFGE), a method developed for separation of large DNA molecules. Whereas standard DNA gel electrophoresis commonly resolves fragments up to approximately 50 kb in size, PFGE fractionates DNA molecules up to 10 Mb. The mechanism driving these separations exploit...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2007.94
更新日期:2007-01-01 00:00:00
abstract::This protocol describes a cell-free system for studying vertebrate centromere and kinetochore formation. We reconstitute tandem arrays of centromere protein A (CENP-A) nucleosomes as a substrate for centromere and kinetochore assembly. These chromatin substrates are immobilized on magnetic beads and then incubated in ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2012.112
更新日期:2012-10-01 00:00:00
abstract::The terminal monosaccharide of cell surface glycoconjugates is typically a sialic acid (SA), and aberrant sialylation is involved in several diseases. Several methodological approaches in sample preparation and subsequent analysis using mass spectrometry (MS) have enabled the identification of glycosylation sites and ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2010.167
更新日期:2010-12-01 00:00:00
abstract::The study of angiogenic endothelial cells (ECs) has in recent years greatly stimulated multiple fields of vascular biology research. A number of cellular models and numerous complex developmental, manipulatory and tumor animal models have been developed to study angiogenesis in vitro and in vivo. To connect the versat...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2009.96
更新日期:2009-01-01 00:00:00
abstract::The pig has emerged as an important large animal model in biomedical and pharmaceutical research. We describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in pigs by using the Sleeping Beauty (SB) transposon system. The protocol is based on co-injection of a plasmid encoding ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2014.010
更新日期:2014-04-01 00:00:00