Monoclonal anti ffa1 antibody rabbit

Cells were grown on coverslips coated with poly-l-lysine, fixed with 4% paraformaldehyde and permeabilized using 0.3% Triton X-100 for 10 min. Afterwards, they were washed with PBS (137 mM NaCl; 2.7 mM KCl; 10 mM NaHPO₄; 2 mM KH₂PO₄), and were then incubated for 1 h in blocking solution (1% Bovine serum albumin plus 5% non-fat milk in PBS) at room temperature, before being incubated overnight with monoclonal anti-FFA1 antibody (rabbit, Abcam) at 4 °C. On the next day, the cells were washed in PBS and then incubated for 2 h with secondary antibody (Alexa488-bound anti-rabbit antibody). Hoescht 33342 (Invitrogen, Waltham, MA, USA ) was used to stain the cell nuclei. Samples were treated with DAKO Fluorescent mounting medium (Sigma) and then visualized using an inverted Olympus FluoView 1000 confocal microscope (GrupoBios S.A, Santiago, Chile).

HT-29 cells cultured in 35 mm dishes were lifted by trypsinization at 37 °C, trypsin activity was stopped by the addition of a complete culture medium, and then cells were pelleted by centrifugation at 600× g for 5 min, and afterwards were fixed by 4% paraformaldehyde for 10 min at 37 °C. Then, they were cooled in ice for 1 min, before being centrifuged at 600× g for 5 min and resuspended using 90% methanol in PBS (137 mM NaCl; 2.7 mM KCl; 10 mM NaHPO₄; 2 mM KH₂PO₄). Cells were then incubated for 30 min at 4 °C, washed twice with PBS, and treated for 60 min with monoclonal anti-FFA1 antibody (rabbit, Abcam, Cambridge, MA, USA) or Isotype control IgG (rabbit, Abcam) at 4 °C. Afterwards, cells were washed twice with PBS and marked with the anti-rabbit Alexa488-bound secondary antibody for 60 min in darkness. Finally, the cells were washed and then resuspended in PBS. Using a FACSCanto II cytometer (BD Biosciences, San Diego, CA, USA), the cells were displayed as plots of forward light scatter versus side light scatter, and the cell population was identified. The mean fluorescence of Alexa488 was determined from a minimum of 10,000 cells using BD FACSDiva 6.1 software (BD Biosciences).