antibod yesebreactivity to mouseisotype of monoclonal antibod y ( clone no. ) gt × hbrachyurynoyesnt * ms × hdppa5yesnt * nt * nd * gt × hgata6noyesnt * gt × hnanogyesdownnogt × hoct 3 / 4yesdownyesgt × hpdx - 1nonoyesgt × hsox17noyesyesms × hcd9yesnominimalmouse igg2b ( clone 209306 ) ms × he - cadherinyesnominimalmouse igg2b ( clone 180224 ) ms × hgata1noyesnt * rat igg2b ( clone 234732 ) ms × hpodxlyesnonomouse igg2a ( clone 222328 ) ms × hsox2yesyesyesmouse igg2a ( clone 245610 ) * nt, not tested; nd, not determined. the expression patterns detected using antibod ies developed in our facility are consistent with data reported using reverse transcriptase-polymerase chain reaction or cdna microarrays. moreover several of the monoclonal antibod ies have differing heavy chain subunits allowing double labeling using subtype specific markers to be performed. in summary, we have developed a useful collection of antibod ies that would be useful for identification of stem cell characteristics and assessment of differentiation. several additional antibod ies to the molecules that have been identified as potential cell lineage markers [ 13 ] are currently under development using the same approach. cloning and expression of brachyury, dppa5, cd9, e - cadherin, gata1, gata6, nanog, oct3 / 4, pdx - 1, podxl, sox2 and sox17brachyury ( aa. 177 – 414 ) were expressed in e. coli and extracellular domains of cd9, e - cadherin, podxl were expressed in mouse nso cells. all proteins were purified and sequenced before they were used as antigens for immunizations and as substrate for antibod y screening and subcloning. production and purification of antibod iesall monoclonal antibod ies were derived from fusions of mouse myeloma with b cells obtained from balb / c mice which had been immunized with purified antigen. the igg fraction of the culture supernatant was purified by protein g affinity chromatography ( sigma ). each panel of antibod ies was screened and selected for their abilities to detect purified recombinant antigen in direct elisa and western blot. all polyclonal antibod ies were derived from sera of goats which had been immunized and boost it with purified antigen. antibod y was purified from the sera by an antigen-affinity chromatography. cells and cell culturehuman caco - 2, mg - 63, mcf - 7, ntera - 2 and mouse d3 cells were purchased from american type culture collection ( atcc ). cells were cultured according to the atcc instructions. information regarding human es cell line hsf - 6 ( nih code uc06 ) can be obtained at the website [ 14 ]. undifferentiated human es cells were cultured according to the protocol provided by the university of california, san francisco in human es culture medium [ dmem supplemented with 20 % knockout serum replacement ( invitrogen ) and 5 ng / ml of bfgf ( r & d systems ) ]. to induce formation of embryoid bodies ( ebs ), es colonies were harvested, separated from the mef feeder cells by gravity, gently resuspended in es culture medium and transferred to non - adherent suspension culture dishes ( corning ). unless otherwise noted, ebs derived from human es cell aggregates were cultured for 8 days in es culture medium deprived of bfgf and used for analysis by immunoh istochemistry as described. western blotcells are solubilized in hot 2× sds gel sample buffer ( 20 mm dithioth reitol, 6 % sds, 0. 25 m tris, ph 6. 8, 10 % glycerol, 10 mm naf and bromophenyl blue ) at 2 × 106 per ml. the extracts are heated in a boiling water bath for 5 minutes and sonicate d with a probe sonicator with 3 – 4 bursts of 5 – 10 seconds each. samples are diluted with 1× sds sample buffer to the desired loading of 1 – 5 × 103 per lane. lysates were resolved by sds - page, transferred to immobilon - p membrane, and immunoblotted with 0. 5 μg / ml primary abs as described in r & d systems website [ 15 ]. immunoh istochemistry antibod ies were used with the appropriate secondary reagents at a concentration of 5 to 10 μg / ml. cells or sections of ebs were fixed with 4 % paraformaldehyde in pbs at room temperature for 20 min, then blocked and permeabilized with 0. 1 % trit on x - 100, 1 % bsa, 10 % normal donkey serum in pbs at room temperature for 45 min. after blocking, cells were incubated with diluted primary antibod y overnight at 4°c followed by coupled anti-mouse or anti-goat igg ( molecular probes ) at room temperature in the dark for an hour. between each step cells were washed with pbs with 0. 1 % bsa. rt - pcrtotal rna was extracted from ebs using trizol ls ( invitrogen ). cdna was synthesized by using superscript ii reverse transcriptase ( invitrogen ) according to the manufacturer ' s recommendations. the pcr primers are available upon request. flow cytometry antibod ies were prepared at the concentration of 0. 1 mg / ml. 10 μl of the stock solution was added to 1 – 2. 5 × 105 cells in a total reaction volume not exceeding 200 μl. the sample was then incubated for 20 min at 2 – 8 °c. following incubation, excess antibod y was removed by washing cells twice with facs buffer ( 2 % fcs and 0. 1 % sodium azide in hank ' s buffer ). after wash, cells were resuspend in 200 μl of facs buffer and the binding of unlabeled monoclonal antibod ies was visualized by adding 10 μl of a 25 μg / ml stock solution of a secondary developing reagent such as goat anti-mouse igg conjugated to a fluorochrome for 20 min at 2 – 8°c. following incubation, cells were washed once with facs buffer, once with pbs. after wash, cells were resuspend in 400 μl of pbs and analyzed on a facscant flow cytometer ( becton - dickinson, mountain view, ca ). five thousand events were collected and analyzed using cell quest software. mapping sites within the genome that are hypersensitive to digestion with dnasei is an important method for identifying dna elements that regulate transcription. the standard approach to locating these dnasei - hypersensitive sites ( dhss ) has been to use southern blotting techniques, although we, and others, have recently published alternative methods using a range of technologies including high - throughput sequencing and genomic array tiling paths. in this article, we describe a novel protocol to use real - time pcr to map dhs. advantages of the technique reported here include the small cell numbers required for each analysis, rapid, relatively low - cost experiments with minimal need for specialist equipment. presented examples include comparative dhs mapping of known tal1 / scl regulatory elements between human embryonic stem cells and k562 cells. mapping the location of dnasei - hypersensitive sites ( dhss ) remains central to developing our understanding of transcriptional regulation. elements with a range of transcriptional regulatory functions have been identified initially as dhss. these include transcriptional enhancers ( 1, 2 ) and repressors ( 3, 4 ) as well as chromatin insulators and barrier elements ( 5, 6 ). a number of techniques have been published recently that permit the mapping of dhss without the need for southern blotting ( 7 – 13 ). these include high - throughput sequencing of cloned dna libraries derived from dnasei - digested chromatin ( 8, 9 ), and a number of different approaches that use genomic tiling path arrays to map dhss ( 7, 11, 13 ). while these approaches have the advantage of covering large genomic regions with a limited number of experiments, they are inherently costly and less applicable to the rapid dhs mapping of specific genomic sites in a range of cell types. an alternative approach that does permit the targeted semi - quantitative dhs mapping of specific loci has used quantitative real - time pcr to map sites from digested dna ( 10 ). this technique depends on the quantification of relative loss of pcr signal observed when pcr primers amplify across regions of digested dna compared with amplification of undigested dna.