Fitc conjugated cd3

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.

Fluorescent‐conjugated anti‐human antibodies including fluorescein isothiocyanate (FITC)‐conjugated CD16 (clone: B73.1), phycoerythrin (PE)‐conjugated CD56 (5.1H11), Peridinin‐chlorophyll‐protein complex (PerCP)‐conjugated NKp44 (clone: P44‐8), Allophycocyanin (APC)‐conjugated CD3 (clone: OKT3), FITC‐conjugated CD3 (clone: OKT3), PerCP‐conjugated CD3 (clone: HIT3a), PerCp‐Cy5 conjugated CXCR3 (clone: G025H7), PerCp‐Cy5‐conjugated NKG2D (clone: 1D11), APC‐conjugated CD11b (clone: ICRF44), PerCP‐Cy5.5–conjugated CD27 (clone: M‐T271), PerCP‐Cy5.5‐conjugated perforin (clone: B‐D48), Alexa Fluor® 647‐conjugated granzyme B (clone: GB11), PerCP‐Cy5.5‐conjugated CD96 (clone: NK92.39) and their cognate isotype controls (all from Biolegend) as well as PerCP‐Cy5.5‐conjugated NKG2C (clone: 134591, R&D system) antibodies were used for immunophenotyping. Lysing solution (BD Biosciences) was used for removing the red blood cells. For washing, fixing, and cell permeabilizing, 1X phosphate buffer saline (PBS), 1% paraformaldehyde solution (Sigma), and 1X perm/wash solution (BD Biosciences) were used, respectively.

The murine splenocytes, TILs and TACs were stained with fluorescein isothiocyanate (FITC)-conjugated CD3 (Biolegend), allophycocyanin (APC)-conjugated CD4 (Biolegend), phycoerythrin (PE)/Cy5.5-conjugated CD8 (Biolegend), PE-conjugated NK1.1 (Biolegend), PE/Cy5.5-conjugated CD19 (Biolegend), APC-conjugated BTLA (CD272) (Biolegend), or PE-conjugated CD223 (eBioscience) for different experiments. Flow cytometric analyses were performed using a BD FACSCalibur flow cytometer (BD Bioscience) with CELLQuest software [23 (link), 24 (link)].