Alexa fluor 488 conjugated anti mouse igg goat polyclonal antibody

HEK293 cells (ATCC) were seeded in 6-well plates at a cell density of 8 × 10⁵ cells/well and cultured for 24 h. The cells were transfected with complex of vector DNA (the FLAG-tagged N188Q ghrelin receptor (control) or the indicated FLAG-tagged ghrelin receptor mutants) and FuGENE HD transfection reagent (Promega). The next day, transfected HEK293 cells were harvested in PBS containing 1 mM EDTA, and then incubated for 30 min on ice with 2.5 μg ml⁻¹ anti-FLAG antibody (Wako) and 5 μg ml⁻¹ Alexa Fluor 488–conjugated anti-mouse IgG goat polyclonal antibody (Thermo Fisher Scientific) in FACS buffer (PBS, 2% FBS, 0.05% NaN₃). Cell-surface expression levels were evaluated by flow cytometry on a Guava EasyCyte Plus Flow Cytometer. FLAG-positive cells were defined as cell populations with signals greater than the top 5% of MOCK cells.

HEK293 cells were seeded in six-well plates at a cell density of 2 × 10⁵ cells/well and cultured for 24 h. The cells were transfected with a complex of vector DNA (the FLAG-tagged MrgD receptor (control) or the indicated FLAG-tagged MrgD receptor mutants) and PEI. The next day, transfected HEK293 cells were harvested in PBS containing 0.53 mM EDTA, and then incubated in D-PBS containing 2% BSA and 2 mM EDTA (blocking buffer) for 30 min on ice. After centrifugation, the cells were stained with 10 μg mL⁻¹ anti-FLAG antibody (FUJIFILM Wako), followed 10 μg mL⁻¹ Alexa Fluor 488–conjugated anti-mouse IgG goat polyclonal antibody (Thermo Fisher Scientific) in blocking buffer. The cells were washed once with D-PBS and resuspended in D-PBS containing 2 mM EDTA. Cell-surface expression levels were evaluated by flow cytometry on an Attune Flow Cytometer (Thermo Fisher Scientific). FLAG-positive cells were defined as cell populations with signals greater than the top 3% of MOCK cells.