Apc labeled anti epcam monoclonal antibody apc epcam

The MDA-MB-231 cell line was obtained from the American Type Culture Collection (Manassas, VA, USA) and was cultured in Dulbecco’s modified Eagle medium (GIBCO, Life Technologies Co., Carlsbad, CA, USA) containing 10% fetal bovine serum harvested using trypsin. EGFR and EpCAM are known to be expressed on the cell membrane of MDA-MB-231 [27 (link),28 (link),29 (link)]; therefore, Alexa Fluor^®488-labeled anti-human EGFR antibody (Alexa^®488-EGFR; Ex: 495 nm, Em: 519 nm; Biolegend, San Diego, CA, USA) and APC-labeled anti-EpCAM monoclonal antibody (APC-EpCAM; Ex: 633 nm, Em: 660 nm; Biolegend, San Diego, CA, USA) were used for immunostaining of the membrane proteins EGFR and CD326 (EpCAM), respectively. The Alexa^®488-EGFR and APC-EpCAM solutions were simultaneously added to the same cell solution in concentrations of 5.0 × 10⁷ molecules/cell and 1.0 × 10⁵ molecules/cell, respectively, and gently mixed for 30 min in a dark place. Then, the sample solution was gently centrifuged at 1500 rpm for 3 min and the supernatant was discarded. The precipitate was finally washed with the medium, and this process was repeated three times.

MCF-7 and MDA-MB-231 cell lines were obtained from the American Type Culture Collection. Both cell lines were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Thermo Fischer Scientific, Waltham, MA, USA) containing 10% fetal bovine serum and antibiotics (100 U/mL penicillin/streptomycin (Gibco), and 250 ng/mL fungizone (Gibco)). The cell lines were then harvested using trypsin.
Alexa Fluor^® 488-labeled antihuman EGFR antibody (488-EGFR; Ex: 495 nm, Em: 519 nm; Biolegend, San Diego, CA, USA) and APC-labeled anti-EpCAM monoclonal antibody (APC-EpCAM; Ex: 633 nm, Em: 660 nm; Biolegend, San Diego, CA, USA) were used to stain the membranes of the membrane proteins, EGFR and CD326 (called EpCAM). EGFR is known to be highly expressed in MDA-MB-231 compared to its expression in MCF-7. EpCAM, on the other hand, is highly expressed in MCF-7 compared to its expression in MDA-MB-231. 488-EGFR and APC-EpCAM solutions were added to all cell solutions in concentrations of 1.0 × 10⁸ and 1.0 × 10⁵ molecules/cell, respectively, and they were gently mixed for 30 min in a dark place. Then, they were followed by gentle centrifugation, and the supernatant was discarded. Cell solutions were finally washed with culture medium. This process was repeated three times.