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. 2014 Feb 1;12(1):55–79. doi: 10.1089/adt.2013.524

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Copyright 2014, Mary Ann Liebert, Inc.

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Fig. 2. — Quantifying IL-6-induced pSTAT3 activation in HNSCC cells by high-content analysis. (A) Grayscale and color composite images of Cal33 cells±IL-6 treatment. Grayscale images of Hoechst 33342-stained nuclei (Ch 1) and pSTAT3-Y705 staining (Ch 2) and color composite images of nonstimulated and IL-6-treated (50 ng/mL, 15 min) Cal33 HNSCC cells. (B) Enlarged grayscale images of Cal33 Hoechst-stained nuclei. Enlarged grayscale images of Hoechst 33342-stained nuclei (Ch 1) of nonstimulated and IL-6 treated (50 ng/mL, 15 min) Cal33 HNSCC cells. (C) Enlarged grayscale images of Cal33 ofpSTAT3-Y705 staining. Enlarged grayscale images of pSTAT3-Y705 staining (Ch 2) from nonstimulated and IL-6-treated (50 ng/mL, 15 min) Cal33 HNSCC cells. (D) Nucleus and cytoplasm masks generated by the TE image analysis module segmentation. Corresponding nucleus (inner, dark green or red) and cytoplasm (outer, light green or red) masks generated by the TE image analysis module segmentation. (E–H) Quantitative data extracted from the digital images of the Hoechst-stained nuclei and pSTAT3-Y705 staining in nonstimulated and IL-6-treated Cal33 HNSCC cells by the TE image analysis module; (E) cell counts per image, (F) average pSTAT3-Y705 intensity in the outer cytoplasm mask area, and (G) average pSTAT3-Y705 intensity in the inner nuclear mask area, (H) average inner:outer (Nuc:Cyt) pSTAT3-Y705 intensity ratio. First, 2,000 Cal33 HNSSC cells were seeded into the wells of 384-well microtiter plates in DMEM containing 10% FBS and cultured overnight in an incubator at 37°C in 5% CO₂ and 95% humidity. The cell culture medium was then aspirated, monolayers were washed once with SFM, fresh SFM was added to the wells, and the plates were returned to the incubator at 37°C in 5% CO₂ and 95% humidity for an additional 24 h. Cal33 cells were treated with SFM (□) or 50 ng/mL of IL-6 (■) for 15 min at 37°C in 5% CO₂ and 95% humidity and were then fixed and stained with Hoechst and a mouse monoclonal anti-p-STAT3-Y705 antibody as described in Materials and Methods (Table 1). Images of two fields of view for two fluorescent channels, Hoechst (Ch 1) and pSTAT3-Y705-FITC (Ch 2), were acquired on the IXU confocal HCS platform using a 20×/0.45NA objective, and the quantitative data presented were extracted from the digital images using the TE image analysis module as described in Materials and Methods. The images presented are representative of similar images obtained in numerous independent experiments, and the data represent the mean±SD of triplicate determinations from one of these independent experiments. Ch 1, channel 1; Ch 2, channel 2; FBS, fetal bovine serum; HCS, high-content imaging; IXU, ImageXpress Ultra; SFM, serum-free tissue culture medium; TE, translocation enhanced; DMEM, Dulbecco's modified Eagle's medium.

Fig. 2. — Quantifying IL-6-induced pSTAT3 activation in HNSCC cells by high-content analysis. (A) Grayscale and color composite images of Cal33 cells±IL-6 treatment. Grayscale images of Hoechst 33342-stained nuclei (Ch 1) and pSTAT3-Y705 staining (Ch 2) and color composite images of nonstimulated and IL-6-treated (50 ng/mL, 15 min) Cal33 HNSCC cells. (B) Enlarged grayscale images of Cal33 Hoechst-stained nuclei. Enlarged grayscale images of Hoechst 33342-stained nuclei (Ch 1) of nonstimulated and IL-6 treated (50 ng/mL, 15 min) Cal33 HNSCC cells. (C) Enlarged grayscale images of Cal33 ofpSTAT3-Y705 staining. Enlarged grayscale images of pSTAT3-Y705 staining (Ch 2) from nonstimulated and IL-6-treated (50 ng/mL, 15 min) Cal33 HNSCC cells. (D) Nucleus and cytoplasm masks generated by the TE image analysis module segmentation. Corresponding nucleus (inner, dark green or red) and cytoplasm (outer, light green or red) masks generated by the TE image analysis module segmentation. (E–H) Quantitative data extracted from the digital images of the Hoechst-stained nuclei and pSTAT3-Y705 staining in nonstimulated and IL-6-treated Cal33 HNSCC cells by the TE image analysis module; (E) cell counts per image, (F) average pSTAT3-Y705 intensity in the outer cytoplasm mask area, and (G) average pSTAT3-Y705 intensity in the inner nuclear mask area, (H) average inner:outer (Nuc:Cyt) pSTAT3-Y705 intensity ratio. First, 2,000 Cal33 HNSSC cells were seeded into the wells of 384-well microtiter plates in DMEM containing 10% FBS and cultured overnight in an incubator at 37°C in 5% CO₂ and 95% humidity. The cell culture medium was then aspirated, monolayers were washed once with SFM, fresh SFM was added to the wells, and the plates were returned to the incubator at 37°C in 5% CO₂ and 95% humidity for an additional 24 h. Cal33 cells were treated with SFM (□) or 50 ng/mL of IL-6 (■) for 15 min at 37°C in 5% CO₂ and 95% humidity and were then fixed and stained with Hoechst and a mouse monoclonal anti-p-STAT3-Y705 antibody as described in Materials and Methods (Table 1). Images of two fields of view for two fluorescent channels, Hoechst (Ch 1) and pSTAT3-Y705-FITC (Ch 2), were acquired on the IXU confocal HCS platform using a 20×/0.45NA objective, and the quantitative data presented were extracted from the digital images using the TE image analysis module as described in Materials and Methods. The images presented are representative of similar images obtained in numerous independent experiments, and the data represent the mean±SD of triplicate determinations from one of these independent experiments. Ch 1, channel 1; Ch 2, channel 2; FBS, fetal bovine serum; HCS, high-content imaging; IXU, ImageXpress Ultra; SFM, serum-free tissue culture medium; TE, translocation enhanced; DMEM, Dulbecco's modified Eagle's medium.