Concentration Assay:Article Title: High-content screening for chemical modulators of embryonal carcinoma cell differentiation and survival.
Article Snippet: Primary and secondary screening assays for compound screening, ntera2 cells were plated at 2500 cells per well of a 96-well μclear flat-bottom plate (greiner Bio-one, stonehouse, UK). each compound was tested at three concentrations (0.1, 1, and 10 μM) and each concentration in triplicate wells. nine wells on each plate contained 0.1% dMso (vehicle) control, and three wells contained 10 μM all-trans retinoic acid as a differentiation control. after 5 days, the cells were washed once with dulbecco’s phosphate-buffered saline (pBs) without Ca2+ and Mg2+ and then fixed with 4% paraformaldehyde for 15 min. this was followed by washing the cells and blocking in pBs supplemented with 10% fCs (blocking buffer) for 1 h. Cells were then incubated with ssea3 primary antibody for 1 h, washed three times in pBs, and incubated with an fitCconjugated secondary antibody for 1 h. Cells in at least three wells on the plate were incubated with secondary antibody only, to determine the background fluorescence. nuclei were counterstained with 10 μg/ml hoechst 33342 (invitrogen). images of stained cells were acquired using an automated microscopy platform (inCell analyzer 1000, ge healthcare, little Chalfont, UK). ten random fields were acquired in each well using a 10× objective. images were analyzed using developer toolbox 1.7 software (ge healthcare). the effects of the hits from the primary screen were confirmed by retesting the compounds at a final concentration of 0.1, 1, 2.5, 5, and 10 μM, using the same protocol as in the primary screen. after 5 days, cells were fixed and stained for pluripotency markers ssea3 and oCt4. ten random fields were acquired in each well using a 10× objective. images were analyzed using developer toolbox 1.7 software.
Control:Article Title: High-content screening for chemical modulators of embryonal carcinoma cell differentiation and survival.
Article Snippet: Primary and secondary screening assays for compound screening, ntera2 cells were plated at 2500 cells per well of a 96-well μclear flat-bottom plate (greiner Bio-one, stonehouse, UK). each compound was tested at three concentrations (0.1, 1, and 10 μM) and each concentration in triplicate wells. nine wells on each plate contained 0.1% dMso (vehicle) control, and three wells contained 10 μM all-trans retinoic acid as a differentiation control. after 5 days, the cells were washed once with dulbecco’s phosphate-buffered saline (pBs) without Ca2+ and Mg2+ and then fixed with 4% paraformaldehyde for 15 min. this was followed by washing the cells and blocking in pBs supplemented with 10% fCs (blocking buffer) for 1 h. Cells were then incubated with ssea3 primary antibody for 1 h, washed three times in pBs, and incubated with an fitCconjugated secondary antibody for 1 h. Cells in at least three wells on the plate were incubated with secondary antibody only, to determine the background fluorescence. nuclei were counterstained with 10 μg/ml hoechst 33342 (invitrogen). images of stained cells were acquired using an automated microscopy platform (inCell analyzer 1000, ge healthcare, little Chalfont, UK). ten random fields were acquired in each well using a 10× objective. images were analyzed using developer toolbox 1.7 software (ge healthcare). the effects of the hits from the primary screen were confirmed by retesting the compounds at a final concentration of 0.1, 1, 2.5, 5, and 10 μM, using the same protocol as in the primary screen. after 5 days, cells were fixed and stained for pluripotency markers ssea3 and oCt4. ten random fields were acquired in each well using a 10× objective. images were analyzed using developer toolbox 1.7 software.
Saline:Article Title: High-content screening for chemical modulators of embryonal carcinoma cell differentiation and survival.
Article Snippet: Primary and secondary screening assays for compound screening, ntera2 cells were plated at 2500 cells per well of a 96-well μclear flat-bottom plate (greiner Bio-one, stonehouse, UK). each compound was tested at three concentrations (0.1, 1, and 10 μM) and each concentration in triplicate wells. nine wells on each plate contained 0.1% dMso (vehicle) control, and three wells contained 10 μM all-trans retinoic acid as a differentiation control. after 5 days, the cells were washed once with dulbecco’s phosphate-buffered saline (pBs) without Ca2+ and Mg2+ and then fixed with 4% paraformaldehyde for 15 min. this was followed by washing the cells and blocking in pBs supplemented with 10% fCs (blocking buffer) for 1 h. Cells were then incubated with ssea3 primary antibody for 1 h, washed three times in pBs, and incubated with an fitCconjugated secondary antibody for 1 h. Cells in at least three wells on the plate were incubated with secondary antibody only, to determine the background fluorescence. nuclei were counterstained with 10 μg/ml hoechst 33342 (invitrogen). images of stained cells were acquired using an automated microscopy platform (inCell analyzer 1000, ge healthcare, little Chalfont, UK). ten random fields were acquired in each well using a 10× objective. images were analyzed using developer toolbox 1.7 software (ge healthcare). the effects of the hits from the primary screen were confirmed by retesting the compounds at a final concentration of 0.1, 1, 2.5, 5, and 10 μM, using the same protocol as in the primary screen. after 5 days, cells were fixed and stained for pluripotency markers ssea3 and oCt4. ten random fields were acquired in each well using a 10× objective. images were analyzed using developer toolbox 1.7 software.
Blocking Assay:Article Title: High-content screening for chemical modulators of embryonal carcinoma cell differentiation and survival.
Article Snippet: Primary and secondary screening assays for compound screening, ntera2 cells were plated at 2500 cells per well of a 96-well μclear flat-bottom plate (greiner Bio-one, stonehouse, UK). each compound was tested at three concentrations (0.1, 1, and 10 μM) and each concentration in triplicate wells. nine wells on each plate contained 0.1% dMso (vehicle) control, and three wells contained 10 μM all-trans retinoic acid as a differentiation control. after 5 days, the cells were washed once with dulbecco’s phosphate-buffered saline (pBs) without Ca2+ and Mg2+ and then fixed with 4% paraformaldehyde for 15 min. this was followed by washing the cells and blocking in pBs supplemented with 10% fCs (blocking buffer) for 1 h. Cells were then incubated with ssea3 primary antibody for 1 h, washed three times in pBs, and incubated with an fitCconjugated secondary antibody for 1 h. Cells in at least three wells on the plate were incubated with secondary antibody only, to determine the background fluorescence. nuclei were counterstained with 10 μg/ml hoechst 33342 (invitrogen). images of stained cells were acquired using an automated microscopy platform (inCell analyzer 1000, ge healthcare, little Chalfont, UK). ten random fields were acquired in each well using a 10× objective. images were analyzed using developer toolbox 1.7 software (ge healthcare). the effects of the hits from the primary screen were confirmed by retesting the compounds at a final concentration of 0.1, 1, 2.5, 5, and 10 μM, using the same protocol as in the primary screen. after 5 days, cells were fixed and stained for pluripotency markers ssea3 and oCt4. ten random fields were acquired in each well using a 10× objective. images were analyzed using developer toolbox 1.7 software.
Incubation:Article Title: High-content screening for chemical modulators of embryonal carcinoma cell differentiation and survival.
Article Snippet: Primary and secondary screening assays for compound screening, ntera2 cells were plated at 2500 cells per well of a 96-well μclear flat-bottom plate (greiner Bio-one, stonehouse, UK). each compound was tested at three concentrations (0.1, 1, and 10 μM) and each concentration in triplicate wells. nine wells on each plate contained 0.1% dMso (vehicle) control, and three wells contained 10 μM all-trans retinoic acid as a differentiation control. after 5 days, the cells were washed once with dulbecco’s phosphate-buffered saline (pBs) without Ca2+ and Mg2+ and then fixed with 4% paraformaldehyde for 15 min. this was followed by washing the cells and blocking in pBs supplemented with 10% fCs (blocking buffer) for 1 h. Cells were then incubated with ssea3 primary antibody for 1 h, washed three times in pBs, and incubated with an fitCconjugated secondary antibody for 1 h. Cells in at least three wells on the plate were incubated with secondary antibody only, to determine the background fluorescence. nuclei were counterstained with 10 μg/ml hoechst 33342 (invitrogen). images of stained cells were acquired using an automated microscopy platform (inCell analyzer 1000, ge healthcare, little Chalfont, UK). ten random fields were acquired in each well using a 10× objective. images were analyzed using developer toolbox 1.7 software (ge healthcare). the effects of the hits from the primary screen were confirmed by retesting the compounds at a final concentration of 0.1, 1, 2.5, 5, and 10 μM, using the same protocol as in the primary screen. after 5 days, cells were fixed and stained for pluripotency markers ssea3 and oCt4. ten random fields were acquired in each well using a 10× objective. images were analyzed using developer toolbox 1.7 software.
Fluorescence:Article Title: High-content screening for chemical modulators of embryonal carcinoma cell differentiation and survival.
Article Snippet: Primary and secondary screening assays for compound screening, ntera2 cells were plated at 2500 cells per well of a 96-well μclear flat-bottom plate (greiner Bio-one, stonehouse, UK). each compound was tested at three concentrations (0.1, 1, and 10 μM) and each concentration in triplicate wells. nine wells on each plate contained 0.1% dMso (vehicle) control, and three wells contained 10 μM all-trans retinoic acid as a differentiation control. after 5 days, the cells were washed once with dulbecco’s phosphate-buffered saline (pBs) without Ca2+ and Mg2+ and then fixed with 4% paraformaldehyde for 15 min. this was followed by washing the cells and blocking in pBs supplemented with 10% fCs (blocking buffer) for 1 h. Cells were then incubated with ssea3 primary antibody for 1 h, washed three times in pBs, and incubated with an fitCconjugated secondary antibody for 1 h. Cells in at least three wells on the plate were incubated with secondary antibody only, to determine the background fluorescence. nuclei were counterstained with 10 μg/ml hoechst 33342 (invitrogen). images of stained cells were acquired using an automated microscopy platform (inCell analyzer 1000, ge healthcare, little Chalfont, UK). ten random fields were acquired in each well using a 10× objective. images were analyzed using developer toolbox 1.7 software (ge healthcare). the effects of the hits from the primary screen were confirmed by retesting the compounds at a final concentration of 0.1, 1, 2.5, 5, and 10 μM, using the same protocol as in the primary screen. after 5 days, cells were fixed and stained for pluripotency markers ssea3 and oCt4. ten random fields were acquired in each well using a 10× objective. images were analyzed using developer toolbox 1.7 software.
Staining:Article Title: High-content screening for chemical modulators of embryonal carcinoma cell differentiation and survival.
Article Snippet: Primary and secondary screening assays for compound screening, ntera2 cells were plated at 2500 cells per well of a 96-well μclear flat-bottom plate (greiner Bio-one, stonehouse, UK). each compound was tested at three concentrations (0.1, 1, and 10 μM) and each concentration in triplicate wells. nine wells on each plate contained 0.1% dMso (vehicle) control, and three wells contained 10 μM all-trans retinoic acid as a differentiation control. after 5 days, the cells were washed once with dulbecco’s phosphate-buffered saline (pBs) without Ca2+ and Mg2+ and then fixed with 4% paraformaldehyde for 15 min. this was followed by washing the cells and blocking in pBs supplemented with 10% fCs (blocking buffer) for 1 h. Cells were then incubated with ssea3 primary antibody for 1 h, washed three times in pBs, and incubated with an fitCconjugated secondary antibody for 1 h. Cells in at least three wells on the plate were incubated with secondary antibody only, to determine the background fluorescence. nuclei were counterstained with 10 μg/ml hoechst 33342 (invitrogen). images of stained cells were acquired using an automated microscopy platform (inCell analyzer 1000, ge healthcare, little Chalfont, UK). ten random fields were acquired in each well using a 10× objective. images were analyzed using developer toolbox 1.7 software (ge healthcare). the effects of the hits from the primary screen were confirmed by retesting the compounds at a final concentration of 0.1, 1, 2.5, 5, and 10 μM, using the same protocol as in the primary screen. after 5 days, cells were fixed and stained for pluripotency markers ssea3 and oCt4. ten random fields were acquired in each well using a 10× objective. images were analyzed using developer toolbox 1.7 software.
Microscopy:Article Title: High-content screening for chemical modulators of embryonal carcinoma cell differentiation and survival.
Article Snippet: Primary and secondary screening assays for compound screening, ntera2 cells were plated at 2500 cells per well of a 96-well μclear flat-bottom plate (greiner Bio-one, stonehouse, UK). each compound was tested at three concentrations (0.1, 1, and 10 μM) and each concentration in triplicate wells. nine wells on each plate contained 0.1% dMso (vehicle) control, and three wells contained 10 μM all-trans retinoic acid as a differentiation control. after 5 days, the cells were washed once with dulbecco’s phosphate-buffered saline (pBs) without Ca2+ and Mg2+ and then fixed with 4% paraformaldehyde for 15 min. this was followed by washing the cells and blocking in pBs supplemented with 10% fCs (blocking buffer) for 1 h. Cells were then incubated with ssea3 primary antibody for 1 h, washed three times in pBs, and incubated with an fitCconjugated secondary antibody for 1 h. Cells in at least three wells on the plate were incubated with secondary antibody only, to determine the background fluorescence. nuclei were counterstained with 10 μg/ml hoechst 33342 (invitrogen). images of stained cells were acquired using an automated microscopy platform (inCell analyzer 1000, ge healthcare, little Chalfont, UK). ten random fields were acquired in each well using a 10× objective. images were analyzed using developer toolbox 1.7 software (ge healthcare). the effects of the hits from the primary screen were confirmed by retesting the compounds at a final concentration of 0.1, 1, 2.5, 5, and 10 μM, using the same protocol as in the primary screen. after 5 days, cells were fixed and stained for pluripotency markers ssea3 and oCt4. ten random fields were acquired in each well using a 10× objective. images were analyzed using developer toolbox 1.7 software.
Software:Article Title: High-content screening for chemical modulators of embryonal carcinoma cell differentiation and survival.
Article Snippet: Primary and secondary screening assays for compound screening, ntera2 cells were plated at 2500 cells per well of a 96-well μclear flat-bottom plate (greiner Bio-one, stonehouse, UK). each compound was tested at three concentrations (0.1, 1, and 10 μM) and each concentration in triplicate wells. nine wells on each plate contained 0.1% dMso (vehicle) control, and three wells contained 10 μM all-trans retinoic acid as a differentiation control. after 5 days, the cells were washed once with dulbecco’s phosphate-buffered saline (pBs) without Ca2+ and Mg2+ and then fixed with 4% paraformaldehyde for 15 min. this was followed by washing the cells and blocking in pBs supplemented with 10% fCs (blocking buffer) for 1 h. Cells were then incubated with ssea3 primary antibody for 1 h, washed three times in pBs, and incubated with an fitCconjugated secondary antibody for 1 h. Cells in at least three wells on the plate were incubated with secondary antibody only, to determine the background fluorescence. nuclei were counterstained with 10 μg/ml hoechst 33342 (invitrogen). images of stained cells were acquired using an automated microscopy platform (inCell analyzer 1000, ge healthcare, little Chalfont, UK). ten random fields were acquired in each well using a 10× objective. images were analyzed using developer toolbox 1.7 software (ge healthcare). the effects of the hits from the primary screen were confirmed by retesting the compounds at a final concentration of 0.1, 1, 2.5, 5, and 10 μM, using the same protocol as in the primary screen. after 5 days, cells were fixed and stained for pluripotency markers ssea3 and oCt4. ten random fields were acquired in each well using a 10× objective. images were analyzed using developer toolbox 1.7 software.
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