Ack red blood cells depletion kit

Leukocytes were isolated from whole blood using ACK red blood cells depletion kit (Leagene, Beijing, China), according to the manufacturer’s protocol. Purified leukocytes were counted and 2 × 10⁵ cells were added to the upper chamber. The supernatant of DLD1/VC or DLD1/circGLIS2 cells was added to the lower chamber to test the chemotaxis effect. After 2 h of incubation, the number of leukocytes translocated to the lower chamber were counted using The Bio-Rad TC20 automated cell counter (Bio-Rad, California, USA). Subsequently, leukocytes obtained from the lower chamber were labeled with FITC conjugated CD3 (Biolegend, California, USA) and APC conjugated CD15 (Biolegend, California, USA) antibodies to test the identity of the leukocytes recruited by the supernatant of CRC cells. The samples were analyzed on FACSCanto Flow Cytometry (BD Biosciences, California, USA) according to the user’s manual.

Three pairs of CRC tissues and matched adjacent noncancerous colorectal tissues were collected from surgical resections at the Sixth Affiliated Hospital, Sun Yat-sen University (Guangdong, China). None of the tissues were exposed to chemo- or radio-therapies prior to surgery. The tissues were frozen and stored in liquid nitrogen until use. Whole blood samples were preserved at 4 °C after collection from healthy donor. The leukocyte were purified by ACK red blood cells depletion kit (Leagene, Beijing, China), according to the manufacturer’s manual. The protocol and the use of clinical materials for research purposes were approved by the Ethics Committee of the Sixth Affiliated Hospital of Sun Yat-sen University (ethical approval number 2018ZSLYEC-008).