Sc 26637

To analyse GLP-1 content, GLUTag cells (1X10 5 cells/ coverslip) were seeded on Geltrex™ coated glass coverslips and cultured overnight until confluent. After treatment with 6.25µg/ml CPE for 24h in standard culture media, cells were permeabilised and fixed by addition of 500µl of ice-cold methanol for 5mins at -20 o C. Non-specific binding was blocked with normal horse serum for 1h at room temperature and cells were then incubated with primary anti-GLP-1 antibody (1:200 dilution in PBS containing 2%BSA) (Goat pAb to GLP-1, SC-26637, Santa Cruz Biotechnology), overnight at 4 o C. Alexa Fluor 594 conjugated donkey anti-goat IgG secondary antibody (1:500 dilution in PBS) (abcam, ab150132) was added to cells and incubated at 37 o C for 1h. Mounting media containing DAPI (Abcam, ab104139) was used to stain the nuclei. Cells were visualised and images captured using an EVOS ® FL fluorescence microscope. Random images were taken from each coverslip and average intensity of 60 cells/coverslip was measured using ZEN lite software. Data was plotted as mean pixel intensity per 1000 pixel 2 .