Pe conjugated mouse anti human nep mab igg

The NEP expression in HOS and Saos-2 cells was determined by flow cytometry after cell staining with PE-conjugated mouse anti-human NEP mAb IgG (BD Biosciences) for 30 min at RT in darkness according to the manufacturer’s recommendations. After washing with PBS, the cells were subjected to analysis on an FACS Calibur (BD Biosciences) equipped with Cell Quest software (BD Biosciences). IgG isotype control antibodies (BD Biosciences) were used as a negative control. The results were presented as the relative mean fluorescence intensity (MFI).

Fluorescence-activated cell sorting (FACS) was performed to quantify the level of NEP in the colon cancer cell lines HT-29, LS 180, SW 948, and SW 620. For this purpose, cells were cultivated in six-well plates in the presence of 10 % FCS for 48 h under standard conditions. The cells were detached with Accutase® Solution, resuspended in a medium with 1 % FCS, and incubated for 60 min under standard conditions. Afterward, cells were centrifuged at 300×g for 5 min and washed with PBS. Then, cells were incubated with phycoerythrin (PE)-conjugated mouse antihuman NEP mAb IgG (BD Biosciences) for 30 min at RT in darkness. In addition to this procedure, a permeabilization step was also incorporated to enable intracellular staining of the cells. After washing with PBS, FACS data, acquired by running the samples on a FACS Calibur (BD Biosciences), were analyzed using Cell Quest software (BD Biosciences). The data were expressed as percent of cells expressing NEP and relative mean fluorescence intensity (MFI). IgG isotype control Ab (BD Biosciences) was used as a negative control.