Guava easycyte 10ht

Flow cytometry was conducted as previously described [29 (link),30 (link)]. Cultured cells were stained using primary antibodies (cetuximab, Cet-IR700, control antibody, Control mAb-IR700) and Alexa Fluor 488-conjugated anti-rat as secondary antibodies. Stained cells were analyzed using Guava easyCyte 10HT (Merck Millipore, Billerica, MA, USA). The data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).

Flow cytometric analysis was performed as previously described^{62 (link), 67} . Alexa Fluor-647 was conjugated to each antibody according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA) as previously described^{34 (link)}. Fluorescent antibody to CD3 (17A2, BioLegend San Diego, CA, USA; or TONBO Biosciences, San Diego, CA, USA), CD4 (GK1.5, eBioscience, San Diego, CA, USA; or BioLegend), CD8 (53–6.7, BioLegend or TONBO), CD127 (SB/199 or eBioRDR5, eBioscience; or R34-R34, TONBO), B220 (RA3–6B2, BioLegend or TONBO), IgM (RMM-1, BioLegend), common-γ (TUGm2, BioLegend) and CRLF2 (FAB5461P, R&D Systems, Minneapolis, MN, USA) were used for cell staining. To detect phospho-STAT5 and phospho-AKT, anti-phospho-STAT5 antibody (SRBCZX, eBioscience) and anti-phospho-AKT antibody (SDRNR, eBioscience) was used respectively. The stained cells were analyzed using a Guava easyCyte 10HT (Merck Millipore Co., Darmstadt, Germany) or Aria flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Dead cells, which were stained using propidium iodide (PI) (Thermo Fisher), were excluded from the analysis. The data were analyzed using the FlowJo program (Tree Star, Ashland, OR, USA).

Flow cytometry was conducted as previously described [3 (link)]. Cultured cells were stained with 0614 mAb or human chimera 0614-5 mAb or IE9 mAb as the primary antibody and Alexa Fluor 488- or 647-conjugated anti-rat or human or mouse-Ig polyclonal antibody (Thermo Fisher Scientific) as the secondary antibody. Stained cells were analyzed on a Guava easyCyte 10HT (Merck Millipore). Dead cells were stained using propidium iodide (PI) (Thermo Fisher Scientific). Data were analyzed using the FlowJo software (Tree Star, Tokyo, Japan).