Anti ifn γ

Tumor tissues were collected, cut into pieces, and then dissolved in radioimmunoprecipitation assay (RIPA) lysis buffer containing protease inhibitor cocktail (Invitrogen, United States). Protein concentration was quantified by a BCA Protein Assay Kit (Solarbio, Beijing, China). Ten micrograms of total protein extract was used for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis. Separated protein in SDS-PAGE gel was transferred onto polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA, United States). After being blocked with 5% skimmed milk for 1 h, the membrane was then incubated with primary antibodies overnight at 4°C: anti-IL10, anti-IL17a, anti-CXCL10, anti-IFN-γ, anti-TNFα, anti-TGF-β1, and anti-TNFR2 antibody (Cell Signaling Technology, MA, United States). The membrane was washed three times with TBST for 5 min each. After being washed, the membrane was further incubated with HRP-linked secondary antibody (1:3,000; #7074; Cell Signaling, MA, United States) at room temperature for 1 h. Then the membrane was washed four times with 1 × TBST, and the protein bands were visualized using an enhanced Chemiluminescence Kit (Santa Cruz Biotechnology, Dallas, TX, United States) and photographed on a gel imager system (Bio-Rad, CA, United States).

Single-cell suspension of 1 × 10⁶ CT-26 and CD44⁺ CT-26 cells (namely, CCSCs) in 95 μl of PBS was incubated with Phycoerythrin (PE)-conjugated CD44 monoclonal antibody (eBioscience, San Diego, CA, USA) in the dark for 30 min at 4˚C and subsequently analyzed using Beckman Coulter FC500 Flow Cytometer with the CellQuest Pro software (BD Biosciences, San Jose, CA, USA) to determine the number of CD44⁺ cells. Similarly, PE-conjugated CD11c monoclonal antibody (eBioscience) was used to determine the number of CD11c⁺ cells in mononuclear cells isolated from bone marrow on day 0 and immature BMDCs from day 7 of culture.
The number of CD80, CD86, MHC-I, and MHC-II cells in unpulsed DCs and DCs pulsed with DRibbles (20 μg/ml) or lysate (20 μg/ml) were determined using anti-CD86, anti-CD80, anti-MHC-I, and anti-MHC-II antibody (all from eBioscience).
The percentages of CD8⁺ IFN-γ⁺ cells in total splenic lymphocytes of each experimental and control group were determined using anti-CD8 (eBioscience) and anti-IFN-γ (Cell signaling technology, Inc., Danvers, MA, USA) antibody.