Acquisition of single cells
Tumor tissue obtained by surgery was enriched by Percoll-based gradient centrifugation. The specific experimental steps were as follows. Fresh tumor samples were fragmented and digested with collagenase IV (Gibco) and DNase I (Sigma) to remove elastase at 37 °C for 30 minutes with stirring. After filtration, washing, and centrifugation, cell fragments and red blood cells in the single cell suspension were removed with Human lymphocyte separation fluid (CL5020; Cedarlane). The obtained single cells were placed in PBS with 1% BSA. The viability and cell counts were determined using a Countess II FL instrument.