cells were simultaneous treated by adding 40 SCFA per well (5 x SCFA stock μl solutions in PBS) using a multi-channel pipette (Labret Biopette) to give a final volume of 200 per well. Treatment concentrations were 0 mM, 1 mM, 5 mM, 10 μl mM, and mM SCFA; the cells were incubated at 37°C for 24 h before being fixed in 4 % formalin for 15 min at room temperature. Untreated samples were included as controls. a) HCT116 cytoskeletal (actin-tubulin) b) Representative plate micrograph plate map Figure 4.1. HCA 96-well plate plan and micrographs of HCT116 cells after treatment with SCFAs. a) Top panel: colour key showing SCFA treatment concentrations; Lower panel: the plate map rows represent triplicate treatments. b) The ICC micrograph shows green fluorescent anti-body staining of cytoskeletal structure and blue (Hoescht) nuclei. The inset represents a single well. [vp, valproic acid; P, propionate; B, butyrate; V, valerate]. 4.4.3. Immunocytochemistry of actin and tubulin cytoskeletal structures After fixing, the nuclei were stained with Hoechst (blue) at a ratio of 1:1250 in digitonin and incubated for 30 min at room temperature. After staining, the cells were washed three times with PBS. The plates were sealed with a sealing film and transported on ice at 4°C to Imagen Biotech, Manchester, where they were stained with Phalloidin for actin and an anti-β tubulin antibody prior to HCA analysis. 4.4.4. HCA quantification of cytoskeletal integrity HCA imaging and analysis was carried out at Imagen Biotech using x10 and x20 objectives and ArrayScan II software (v. 3.5.1.2). Hoechst nuclear staining enabled Regions of Interest (ROIs) per well to be identified and corrections to background fluorescence to be made. The total staining intensity
cells were simultaneous treated by adding 40 μl SCFA per well (5 x SCFA stock solutions in PBS) using a multi-channel pipette (Labret Biopette) to give a final volume of 200 μl per well. Treatment concentrations were 0 mM, 1 mM, 5 mM, 10 mM, and mM SCFA; the cells were incubated at 37°C for 24 h before being fixed in 4 % formalin for 15 min at room temperature. Untreated samples were included as controls. a) HCT116 cytoskeletal (actin-tubulin) plate map b) Representative plate micrograph Figure 4.1. HCA 96-well plate plan and micrographs of HCT116 cells after treatment with SCFAs. a) Top panel: colour key showing SCFA treatment concentrations; Lower panel: the plate map rows represent triplicate treatments. b) The ICC micrograph shows green fluorescent anti-body staining of cytoskeletal structure and blue (Hoescht) nuclei. The inset represents a single well. [vp, valproic acid; P, propionate; B, butyrate; V, valerate]. 4.4.3. Immunocytochemistry of actin and tubulin cytoskeletal structures After fixing, the nuclei were stained with Hoechst (blue) at a ratio of 1:1250 in digitonin and incubated for 30 min at room temperature. After staining, the cells were washed three times with PBS. The plates were sealed with a sealing film and transported on ice at 4°C to Imagen Biotech, Manchester, where they were stained with Phalloidin for actin and an anti-β tubulin antibody prior to HCA analysis. 4.4.4. HCA quantification of cytoskeletal integrity HCA imaging and analysis was carried out at Imagen Biotech using x10 and x20 objectives and ArrayScan II software (v. 3.5.1.2). Hoechst nuclear staining enabled Regions of Interest (ROIs) per well to be identified and corrections to background fluorescence to be made. The total staining intensity
PhD_Thesis_%28KilnerJul13%29.docx