Alexa fluor 647 anti mouse cd326 epcam

LAP (laponite-FN) was provided by BYK Netherlands B.V. (Deventer, Netherlands). Poly (ethylene glycol) 2000 (PEG); methoxy poly (ethylene glycol) 2000 (mPEG); DOX·HCl were obtained from AvaChem Scientific (San Antonio, TX, USA). 4-Nitrophenyl chloroformate; 1,3-diamino-2-propanol (Dimethylamino) pyridine (DMAP); N,N-diisopropylethylamine (DiPEA); 3-aminopropyldimethylethoxysilane (APMES); sodium hydroxide; hydrochloric acid and XTT reagents were all purchased from Sigma-Aldrich (St. Louis, MO, USA). A549 cells were purchased from Japanese Collection of Research Bioresources (JRCB) Cell Bank (Tokyo, Japan). Trypsin-EDTA 0.25%, 7-aminoactinomycin (7-AAD), Fixable Viability Dye eFluor™ 450 were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Alexa Fluor^® 647 anti-mouse CD326 (EpCAM) was purchased from BioLegend (San Diego, CA, USA). All solvents were purchased from Sigma-Aldrich and used as received. Deionized (DI) water was used in all the experiments. Dialysis membranes were purchased from Spectrum Laboratories (Rancho Dominquez, CA, USA).

Embryonic day 16.5 thymuses and skins, and day five post birth skins were digested in 1 mg/mL collagenase (Wako, 037-17603) in DMEM (Nacalai Tesque, 08459-64) at 37 °C for 15 min, and then in 0.25% trypsin in 1 mM EDTA-PBS at 37 °C for 5–10 min. After centrifuging, cell pellets were resuspended to Feeder Medium and filtered through a 37-µm mesh. Samples were stained with Alexa Fluor 647 anti-mouse CD326 (Ep-CAM) (1:100, Biolegend 118212) and PerCP anti-mouse CD45 (1:100, Biolegend 103130) at 4 °C for 30 min. An MA900 Multi-Application Cell Sorter (Sony) was used to collect Epcam-positive and CD45-negative cells (10,000 cells/batch).
Since the E16.5 thymic samples were small in size and composed of a low number of thymic epithelium cells, five thymuses were pooled together as one batch.