9). The use of CHO-ldlD cells for flow cytometric analysis is complicated by the culture characteristics of the CHO-ldlD cells and therefore needed optimization. Since the CHO-ldlD cells scavenge the medium for free Gal and GalNAc, they must be cultured at low serum concentrations, to preserve the glycosylation defect. Additionally, because CHO-ldlD cells are adherent, the generation of a single cell suspension is accompanied by cell death. Dead cells are responsible

for a specific binding of antibodies and co-staining with 7AAD showed that in the 7AAD positive population there is a subpopulation TSA HDAC in vivo clearly positive for the MUC1 antibodies ( Fig. 3A). Moreover, reactivity with isotype antibody control could be detected, confirming the aspecific staining of the 7AAD positive cells. To decrease the number of dead cells and increase the yield, two different harvesting techniques were evaluated. Cell scraping was compared with trypsinization of the CHO-ldlD MUC1 cells. In contrast to cell scraping, trypsinization reduced the number of dead cells Atezolizumab mw by 30%, however flow cytometric analysis showed that trypsinization induced expression of new epitiopes

reactive with the MUC1 specific antibodies, but not isotype control antibody ( Fig. 3B). Cleaving of the MUC1 peptide by trypsin could be responsible for this new epitope formation. Even though cell scraping induces a lot of cell death, it remains Methane monooxygenase the preferred option in this system since dead cells can be excluded using 7AAD staining. As shown in Fig. 2, the CHO-ldlD MUC1 system is effective in generating MUC1-Tn epitopes. To analyse if MUC1-Tn antibodies are present in sera of breast cancer patients as well as in healthy controls, CHO-ldlD MUC1 cells were cultured in the presence or absence of GalNAc and prior to flowcytometric analysis

cells were incubated with human serum. The CHO-ldlD cells were taken along as a negative control, to exclude aspecific or specific reactivity. Secondary antibody staining was performed for detection of serum antibodies to MUC1 and MUC1-Tn. Both anti-human IgM- and IgG-detecting secondary antibodies were used to discriminate between primary (IgM) and secondary humoral responses (IgG). Healthy controls as well as breast cancer patients did not show specific binding of serum antibodies with CHO-ldlD MUC1 cells cultured with or without GalNAc. Eventhough in the serum of breast cancer patients repetitively a marginal shift of the histogram could be observed when a secondary IgM-recognizing antibody was used, this shift did not reach significance ( Fig. 4A).