Analysis of tumor associated transmembrane enzyme CAIX in HT-29 cells by flow cytometry

Abstract

Carbonic anhydrase IX (CAIX) is a transmembraneenzyme associated with tumor metabolism. Several studies impli-cated that CAIX could be a prognostic marker in several type oftumor. Therefore, the detection of protein is valuable for clinicalstudies. In this study, the protein expression of Carbonic anhydr-ase IX was analysed by flow cytometry in adherent colon carci-noma cells, namely HT-29. In order to optimise the detection ofHT-29 cells, several parametres have been assessed. Sample prep-aration that means produce a suspension of single cells is a veryimportant experimental stage in flow cytometry. Two differentmethods was used for the preparation of cells to flow cytometry,namely chemical methods Tripsin-EDTA and physical method,cell scrabers tecniques. PE staining Human CAIX monoclonalantibody was used for the detection. . According to the resultsobtained from this optimization study, cell suspension prepara-tion by cell scraper resulted in the more significantly CAIX pro-tein expression in HT-29 cells than chemical Tripsin-EDTAmethod. This could be that CAIX that is a membrane protein islikely to being damaged by Tripsin-EDTA method during thetreatment.