Flow cytometry

We used CountBright™ absolute counting beads (C36950; Invitrogen) to calculate the number of various immune cell subsets. CountBright™ absolute counting beads are a calibrated suspension of microspheres that are brightly fluorescent across a wide range of excitation and emission wavelengths, and can be used with most cell types. Counting beads were gently vortexed for 30 seconds, and then immediately added to the stained cell suspension. The well mixed cell suspension was assayed via flow cytometry. By comparing the ratio of bead events to cell events, absolute numbers of cells in the sample can be calculated. 

All samples used for flow cytometry analysis were stained fresh. As a consequence of the storage condition and processing time of samples directly affects cell viability and staining quality, immediately upon sacrifice, fresh samples are stored on ice during the whole experiment. In general, for serous tumor tissues, such as B16-F10 tumor mass, we would just mechanically grind it through a 70-µm cell strainer in PBS with 2% FBS. And when the tumor tissues were solid, such as MC38 tumor mass, we would firstly minced tissues into small pieces, then digested with 1 mg/mL collagenase IV (Sigma-Aldrich) and 0.2 mg/mL DNase I (Sigma-Aldrich) in serum free media for 30 minutes at 37°C. Cells were filtered through 40-µm nylon strainers (Thermo Fisher Scientific), and washed twice in PBS with 2% FBS. Red blood cells were then removed with red blood cell lysis buffer (Sigma-Aldrich). After completing the above steps for a maximum of 2 hours, we started the staining process.