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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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CellFinder
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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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CellFinder
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A) Immunostaining with goat anti-human SOX17 (Red), is contrasted with Fluoro Nissl nuclear staining (Green). (
|
[] |
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B) Immunostaining with goat anti-human GATA6 (Red), is contrasted with DAPI nuclear staining (Blue). (
|
[] |
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C) Immunostaining with goat anti-human brachyury (Red), is contrasted with DAPI nuclear staining (Blue). (
|
[] |
CellFinder
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D) Immunostaining with mouse anti-human GATA1 (Red).
|
[] |
CellFinder
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Note that each antibody recognizes subsets of EB cells.
|
[
{
"end": 54,
"label": "CellType",
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"text": null
}
] |
CellFinder
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Scale bars = 100 μm. (
|
[] |
CellFinder
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E) The differentiation status of EB is detected by RT-PCR using different germ layer cell markers.
|
[
{
"end": 89,
"label": "CellType",
"start": 74,
"text": null
},
{
"end": 84,
"label": "Tissue",
"start": 74,
"text": null
}
] |
CellFinder
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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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{
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{
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{
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] |
CellFinder
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G3PDH was a positive control showing similar amount of RNA samples were used for analysis.
|
[] |
CellFinder
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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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{
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{
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{
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{
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{
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{
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},
{
"end": 57,
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}
] |
CellFinder
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Cross-reactivity to mouse of goat anti-Oct3/4, goat anti-PDX-1, goat anti-SOX17 and mouse anti-SOX2 was detected.
|
[] |
CellFinder
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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
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Goat anti-Nanog and mouse anti-PODXL antibodies appear to be human-specific as well (data not shown).
|
[] |
CellFinder
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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.
|
[
{
"end": 84,
"label": "CellType",
"start": 82,
"text": null
}
] |
CellFinder
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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
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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
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Moreover several of the monoclonal antibodies have differing heavy chain subunits allowing double labeling using subtype specific markers to be performed.
|
[] |
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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
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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
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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
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1–116), GATA1 (a.a.
|
[] |
CellFinder
|
1–413), GATA6 (aa.
|
[] |
CellFinder
|
1–449), Nanog (aa.
|
[] |
CellFinder
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153–305), Oct3/4 (aa.
|
[] |
CellFinder
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1–265), PDX-1 (aa.
|
[] |
CellFinder
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1–283), SOX2 (aa.
|
[] |
CellFinder
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135–317) and SOX17 (aa.
|
[] |
CellFinder
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177–414) were expressed in E. Coli and extracellular domains of CD9, E-Cadherin, PODXL were expressed in mouse NSO cells.
|
[
{
"end": 120,
"label": "CellType",
"start": 105,
"text": null
},
{
"end": 120,
"label": "CellType",
"start": 111,
"text": null
}
] |
CellFinder
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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
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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.
|
[
{
"end": 122,
"label": "CellType",
"start": 115,
"text": null
},
{
"end": 109,
"label": "Tissue",
"start": 96,
"text": null
}
] |
CellFinder
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The IgG fraction of the culture supernatant was purified by Protein G affinity chromatography (Sigma).
|
[] |
CellFinder
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Each panel of antibodies was screened and selected for their abilities to detect purified recombinant antigen in direct ELISA and Western blot.
|
[] |
CellFinder
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All polyclonal antibodies were derived from sera of goats which had been immunized and boost it with purified antigen.
|
[] |
CellFinder
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Antibody was purified from the sera by an antigen-affinity chromatography.
|
[] |
CellFinder
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Cells and cell cultureHuman Caco-2, MG-63, MCF-7, NTERA-2 and mouse D3 cells were purchased from American Type Culture Collection (ATCC).
|
[
{
"end": 34,
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{
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"label": "CellType",
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},
{
"end": 70,
"label": "CellLine",
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},
{
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},
{
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"text": null
},
{
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"label": "CellLine",
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}
] |
CellFinder
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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].
|
[
{
"end": 34,
"label": "CellType",
"start": 28,
"text": null
},
{
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"label": "CellLine",
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"text": null
},
{
"end": 61,
"label": "CellLine",
"start": 57,
"text": null
}
] |
CellFinder
|
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)].
|
[
{
"end": 31,
"label": "CellType",
"start": 23,
"text": null
},
{
"end": 139,
"label": "CellType",
"start": 137,
"text": null
}
] |
CellFinder
|
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).
|
[
{
"end": 212,
"label": "Tissue",
"start": 205,
"text": null
},
{
"end": 57,
"label": "Tissue",
"start": 49,
"text": null
},
{
"end": 38,
"label": "Tissue",
"start": 23,
"text": null
},
{
"end": 43,
"label": "Tissue",
"start": 40,
"text": null
},
{
"end": 48,
"label": "CellType",
"start": 46,
"text": null
},
{
"end": 96,
"label": "CellType",
"start": 93,
"text": null
},
{
"end": 146,
"label": "CellType",
"start": 144,
"text": null
},
{
"end": 109,
"label": "CellType",
"start": 97,
"text": null
},
{
"end": 154,
"label": "Tissue",
"start": 147,
"text": null
}
] |
CellFinder
|
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.
|
[
{
"end": 27,
"label": "Tissue",
"start": 24,
"text": null
},
{
"end": 65,
"label": "Tissue",
"start": 55,
"text": null
},
{
"end": 96,
"label": "CellType",
"start": 94,
"text": null
}
] |
CellFinder
|
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.
|
[
{
"end": 24,
"label": "Tissue",
"start": 21,
"text": null
}
] |
CellFinder
|
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).
|
[
{
"end": 38,
"label": "Tissue",
"start": 35,
"text": null
}
] |
CellFinder
|
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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