Polyclonal fluorescein isothiocyanate fitc conjugated goat anti mouse igg and igm specific antibody

For the determination of ceramide, a monoclonal antibody-based assay was used. To this end, a 100 µl suspension of erythrocytes was stained for 1 hour at 37°C with 1 µg/ml anti ceramide antibody (clone MID 15B4, Alexis, Grünberg, Germany) in PBS containing 0.1% bovine serum albumin (BSA) at a dilution of 1:10. The samples were washed twice with PBS-BSA. The cell suspension was subsequently stained for 30 minutes with polyclonal fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG and IgM specific antibody (Pharmingen, Hamburg, Germany) diluted 1:50 in PBS-BSA. Unbound secondary antibody was removed by repeated washing with PBS-BSA. The samples were analyzed by flow cytometric analysis with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. As a control, secondary antibody alone was used. Finally, the geomean of the ceramide-dependent fluorescence was determined.

For the determination of ceramide, a monoclonal antibody-based assay was used. To this end, a 100 µl suspension of erythrocytes was stained for 1 hour at 37°C with 1 µg/ml anti ceramide antibody (clone MID 15B4, Alexis, Grünberg, Germany) in PBS containing 0.1% bovine serum albumin (BSA) at a dilution of 1:10. The samples were washed twice with PBS-BSA. The cell suspension was subsequently stained for 30 minutes with polyclonal fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG and IgM specific antibody (Pharmingen, Hamburg, Germany) diluted 1:50 in PBS-BSA. Unbound secondary antibody was removed by repeated washing with PBS-BSA. The samples were analyzed by flow cytometric analysis with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. Finally, the geomean of the ceramide-dependent fluorescence was determined.