Alexa fluor 488 conjugated igg secondary antibody

M3814 was kindly provided by ChemieTek (Indianapolis, IN, USA). FBS, penicillin/streptomycin, DMEM, and trypsin EDTA were purchased from Corning Incorporated (Corning, NY, USA). mitoxantrone, [³H] (2.5 Ci/mmol) was purchased from Moravek Biochemicals, Inc. (Brea, CA, USA). The Alexa Fluor 488 conjugated IgG secondary antibody and GAPDH monoclonal antibody (GA1R) (catalog number MA5-15738) were obtained from Thermo Fisher Scientific Inc (Rockford, IL, USA). Primary antibody against ABCG2, clone BXP-21 (catalog number MAB4146), was obtained from Millipore (Billerica, MA, USA). Anti-mouse IgG, HRP-linked antibody (catalog number 7076S) was purchased from Cell Signaling Technology Inc (Danvers, MA, USA). PBS and BSA were purchased from VWR chemicals, LLC (Solon, OH, USA). Ko143 was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Paraformaldehyde, triton X-100, 4',6-diamidino-2-phenylindole (DAPI), mitoxantrone, doxorubicin, vincristine, verapamil, cisplatin, methylthiazolyldiphenyl-tetrazolium bromide (MTT), DMSO, and all other chemicals were requested from Sigma Chemical Co (St. Louis, MO, USA).

The cellular localization of P-gp and BCRP were visualized using immunofluorescence staining (Wang et al., 2020 (link)). Briefly, the cells were plated at a density of 10,000 cells per well in a 24-well plate format. After cell attachment, different conditions were applied to the designated wells. After 72 h treatment, cells were fixed, permeabilized, and blocked with 5% bovine serum albumin (BSA). Antibodies against P-gp (MA1-26528, Thermo Fisher Scientific) or BCRP (MAB4155, Thermo Fisher Scientific) were incubated with the cells overnight at 4°C. The cells were then incubated with Alexa Fluor 488 conjugated IgG secondary antibody (A-11001, Thermo Fisher Scientific, Waltham, MA) and further counterstained with DAPI. Immunofluorescence images were captured by a fluorescence microscope.