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These results suggest that both endodermal and mesodermal precursors exist in EBs with FGF withdrawal for 8 days. | [
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However, no PDX-1-positive cells were seen in EBs differentiated with the same treatment (data not shown).Figure 4Differentiated EBs were analyzed by either immunocytochemistry or RT-PCR to the indicated molecular markers. ( | [
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A) Immunostaining with goat anti-human SOX17 (Red), is contrasted with Fluoro Nissl nuclear staining (Green). ( | [] | CellFinder |
B) Immunostaining with goat anti-human GATA6 (Red), is contrasted with DAPI nuclear staining (Blue). ( | [] | CellFinder |
C) Immunostaining with goat anti-human brachyury (Red), is contrasted with DAPI nuclear staining (Blue). ( | [] | CellFinder |
D) Immunostaining with mouse anti-human GATA1 (Red). | [] | CellFinder |
Note that each antibody recognizes subsets of EB cells. | [
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Scale bars = 100 μm. ( | [] | CellFinder |
E) The differentiation status of EB is detected by RT-PCR using different germ layer cell markers. | [
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Selected endoderm markers AFP, FoxA2; mesoderm markers Hand1, MSX1 and ectoderm marker Msl1 were all highly expressed in the EB samples while their expression was either undetectable or at low level in the ES samples. | [
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G3PDH was a positive control showing similar amount of RNA samples were used for analysis. | [] | CellFinder |
Examination of cross-reactivity of antibodies on mouse ES and differentiated cellsWe have also examined the cross-reactivities of these antibodies to mouse ES cells using mouse D3 ES cell line and mouse fetal endodermal tissue. | [
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... | CellFinder |
Cross-reactivity to mouse of goat anti-Oct3/4, goat anti-PDX-1, goat anti-SOX17 and mouse anti-SOX2 was detected. | [] | CellFinder |
Minimal cross-reactivity to mouse, measured by 10% intensity to human by higher than control cells, was observed in mouse anti-CD9 and mouse anti-E-cadherin antibodies. | [] | CellFinder |
Goat anti-Nanog and mouse anti-PODXL antibodies appear to be human-specific as well (data not shown). | [] | CellFinder |
The subtypes of monoclonal antibodies were also identified in the best clones. | [] | CellFinder |
These results are summarized in Table 2.Table 2Summary of antibodies detection in ES and EB samples. | [
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AntibodyESEBReactivity to mouseIsotype of monoclonal antibody (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. | [] | CellFinder |
The expression patterns detected using antibodies developed in our facility are consistent with data reported using reverse transcriptase-polymerase chain reaction or cDNA microarrays. | [] | CellFinder |
Moreover several of the monoclonal antibodies have differing heavy chain subunits allowing double labeling using subtype specific markers to be performed. | [] | CellFinder |
In summary, we have developed a useful collection of antibodies that would be useful for identification of stem cell characteristics and assessment of differentiation. | [] | CellFinder |
Several additional antibodies to the molecules that have been identified as potential cell lineage markers [13] are currently under development using the same approach. | [] | CellFinder |
Cloning and expression of Brachyury, DPPA5, CD9, E-Cadherin, GATA1, GATA6, Nanog, Oct3/4, PDX-1, PODXL, SOX2 and SOX17Brachyury (aa. | [] | CellFinder |
1–202), DPPA5 (a.a. | [] | CellFinder |
1–116), GATA1 (a.a. | [] | CellFinder |
1–413), GATA6 (aa. | [] | CellFinder |
1–449), Nanog (aa. | [] | CellFinder |
153–305), Oct3/4 (aa. | [] | CellFinder |
1–265), PDX-1 (aa. | [] | CellFinder |
1–283), SOX2 (aa. | [] | CellFinder |
135–317) and SOX17 (aa. | [] | CellFinder |
177–414) were expressed in E. Coli and extracellular domains of CD9, E-Cadherin, PODXL were expressed in mouse NSO cells. | [
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All proteins were purified and sequenced before they were used as antigens for immunizations and as substrate for antibody screening and subcloning. | [] | CellFinder |
Production and purification of antibodiesAll monoclonal antibodies were derived from fusions of mouse myeloma with B cells obtained from BALB/c mice which had been immunized with purified antigen. | [
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The IgG fraction of the culture supernatant was purified by Protein G affinity chromatography (Sigma). | [] | CellFinder |
Each panel of antibodies was screened and selected for their abilities to detect purified recombinant antigen in direct ELISA and Western blot. | [] | CellFinder |
All polyclonal antibodies were derived from sera of goats which had been immunized and boost it with purified antigen. | [] | CellFinder |
Antibody was purified from the sera by an antigen-affinity chromatography. | [] | CellFinder |
Cells and cell cultureHuman Caco-2, MG-63, MCF-7, NTERA-2 and mouse D3 cells were purchased from American Type Culture Collection (ATCC). | [
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Cells were cultured according to the ATCC instructions. | [] | CellFinder |
Information regarding human ES cell line HSF-6 (NIH code UC06) can be obtained at the website [14]. | [
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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)]. | [
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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). | [
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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 immunohistochemistry as described. | [
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Western blotCells are solubilized in hot 2× SDS gel sample buffer (20 mM dithiothreitol, 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 sonicated with a probe sonicator with 3–4 bursts of 5–10 seconds each. | [] | CellFinder |
Samples are diluted with 1× SDS sample buffer to the desired loading of 1–5 × 103 per lane. | [] | CellFinder |
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]. | [] | CellFinder |
ImmunohistochemistryAntibodies were used with the appropriate secondary reagents at a concentration of 5 to 10 μg/ml. | [] | CellFinder |
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% Triton X-100, 1% BSA, 10% normal donkey serum in PBS at room temperature for 45 min. | [
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After blocking, cells were incubated with diluted primary antibody overnight at 4°C followed by coupled anti-mouse or anti-goat IgG (Molecular Probes) at room temperature in the dark for an hour. | [] | CellFinder |
Between each step cells were washed with PBS with 0.1% BSA. | [] | CellFinder |
RT-PCRTotal RNA was extracted from EBs using Trizol LS (Invitrogen). | [
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cDNA was synthesized by using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's recommendations. | [] | CellFinder |
The PCR primers are available upon request. | [] | CellFinder |
Flow cytometryAntibodies 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. | [] | CellFinder |
Following incubation, excess antibody was removed by washing cells twice with FACS buffer (2% FCS and 0.1% sodium azide in Hank's buffer). | [] | CellFinder |
After wash, cells were resuspend in 200 μL of FACS buffer and the binding of unlabeled monoclonal antibodies 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. | [] | CellFinder |
Following incubation, cells were washed once with FACS buffer, once with PBS. | [] | CellFinder |
After wash, cells were resuspend in 400 μL of PBS and analyzed on a FACScant flow cytometer (Becton-Dickinson, Mountain View, CA). | [] | CellFinder |
Five thousand events were collected and analyzed using CELL Quest software. | [] | CellFinder |
[] | CellFinder |
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