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.