Emc001b

Bone marrow-derived dendritic cells (BMDCs) were isolated from the bone marrow after 8 ~ 10 days of differentiation with granulocyte-macrophage colony-stimulating factor (20 ng/mL, Sino Biological) and IL-4 (10 ng/mL, Sino Biological). Macrophages were purified from tumor tissues with Anti-F4/80 MicroBeads (130-110-443, Miltenyi Biotech Inc.). BMDCs and macrophages were stained with the CFSE Cell Division Tracker Kit (423801, BioLegend). CT26-RFP cells were treated with drugs as indicated for 4 h and then added to the CFSE-labeled BMDC or macrophage culture at a 1:1 ratio. For flow cytometry, the cells were harvested and tested on a NovoCyte flow cytometer. BMDCs and macrophages that phagocytosed CT26 cells were CFSE and RFP-double-positive. For immunofluorescence, cells were fixed in 4% paraformaldehyde. Cell nuclei were stained with DAPI. The concentrations of IL-1β (EMC001b, Neobioscience), IL-12 (EMC006, Neobioscience), IL-18 (EMC011, Neobioscience), and CXCL9 (EK0733, BOSTER) in the supernatant after 24 h of co-culture were quantified using the ELISA Kit.

Collect CKD mouse serum and RAW264.7 cell culture supernatant. The concentration of IL-1β (Neobioscience, EMC001b), IL-6 (Neobioscience, EMC004), and TNF-α (Neobioscience, 500850) in serum and culture medium were detected according to the instructions of the ELISA kit. The absorbance value of each well was measured at 450 nm by BioTeK reader.

Level of IL-1β (EMC001b, Neobioscience), IL-6 (EMC004, Neobioscience), IL-10 (EMC005, Neobioscience) and TNF-α (EMC102a, Neobioscience) in cell supernatants were measured. And CHIL3 levels in serum were measured using an ELISA kit (ELM-YM1, RayBio, GA, USA). According to the manufacturer's instructions. Added samples and standard into strips and incubate for 2.5 hours at room temperature with gentle shaking. Discard the solution and washed. Added 100 µl of prepared biotinylated antibody for 1 hour. Then reacted with prepared Streptavidin solution. TMB One-Step Substrate Reagent used. 45 minutes after chromogenic reaction, stop reaction was added and each well read at 450 nm immediately.

The levels of the inflammatory cytokine TNF-α, IL-1β and IL-10 in cell supernatants were measured using ELISA kits (cat. nos. EMC102a, EMC001b and EMC005, respectively; Neobioscience Technology Co., Ltd.) according to the manufacturer’s instructions.

Cytokine levels in serum or conditioned media were detected using an ELISA kit following the manufacturer’s instructions, including LXA4, tumor necrosis factor alpha (TNF-α) (EMC102a; NeoBioScience, Shenzhen, China) and IL-1β (EMC001b; NeoBioScience, Shenzhen, China).

After the indicated treatments, the cell supernatant of each group was collected in a 1.5 ml EP tube, and the production of TNF‐α and IL‐1β was measured. Commercially available ELISA kits for TNF‐α (Neobioscience, EMC102a) and IL‐1β (Neobioscience, EMC001b) were utilized to quantify the levels of these cytokines according to the manufacturer's instructions. The optical density at 450 nm was measured by using a multifunctional enzyme labelling instrument (SpectraMax i3, Afghanistan).