Fix perm kit

PBLs without treatment, PBLs stimulated with pamidronate, and PBLs cocultured with auto- or allo- DCs^pami+ were treated with phorbol myristate acetate (PMA) and ionomycin Cocktail (Thermo Fisher Scientific) at 37°C for 4 h. Cells were collected, fixed and permeabilized with the FIX&PERM kit (MultiSciences Biotech, China) according to the manufacturer's instructions, and stained with PE anti-ki67, BV421 anti-IFN-γ, and PerCP anti-TNF-α antibodies (Biolegend, USA).

For detection of intracellular cytokines, cocultured cells were treated with 1x Protein Transport Inhibitor Cocktail (500x, eBioscience, USA) for 5 h at 37°C. Following staining with anti-CD4, anti-CD8 and anti-TCRαβ, cells were fixed and permeabilized with the FIX&PERM kit (MultiSciences Biotech, China), and then were stained with anti-TNF-α, anti-IFN-γ and anti-IL-17 antibodies. Besides, positive controls were included to demonstrate that the cytokine staining was appropriate for use in our study. PBMCs were freshly isolated from healthy donors and treated with Cell Stimulation Cocktail (plus protein transport inhibitors) (500x, eBioscience, USA) for 5 h and collected for detection of intracellular cytokines.

Materials: renal clear cell carcinoma cell line 786-O, normal renal cell line HK-2, THP-1 cells, fetal bovine serum (Procell, Wuhan, China), 1640 medium, DMEM medium (meilunbio, Dalian, China), 0.1% crystal violet dye, collagenase IV, hyaluronidase, DNA enzyme, Ficoll paque plus (Solarbio, Beijing, China), 8 μ m and 0.4 μ m transwell chambers, matrix glue (Corning, New York, USA). CD68, CD86 and CD206 flow antibodies, IL-4 and IL-10 (Biolegend, Beijing, China), primers (Sangon Biotech, Shanghai, China), PMA (MedChemExpress, New Jersey, USA), FIX&PERM Kit (MULTISCIENCES, Hangzhou, China). Reverse transcription Kit and qPCR Kit (TransGen Biotech, Beijing, China).

iNK cells were incubated with or without PMA/ionomycin (MULTISCIENCES), K562 tumor cells (E:T = 1:2), or recombinant human IL-2 (1000 IU/mL, Miltenyi) for 2 h, followed by adding BFA/Monensin (MULTISCIENCES) for additional 2-h incubation. Cells were stained with anti-human CD45 (Biolegend, HI30), CD3 (Biolegend, HIT3a), and CD56 (Biolegend, HCD56) antibodies. FIX & PERM Kit (MULTISCIENCES) was used for fixation and permeabilization, followed by intracellular staining for IFN-γ (Biolegend, 4 S.B3) or TNF-α (Biolegend, MAb11). The cells were analyzed with BD LSRFortessa X-20 cytometer (BD Biosciences).

The purified spleen and liver cells were counted and their viability assessed using the trypan blue exclusion method.²⁹ Then, the absolute cell counts (2 × 10⁶) and suspension were in the complete RPMI 1640 medium. All cells were detected by BD FACSVerse and analysed by FlowJo software (Treestar, Inc, San Carlos, USA).
For Th1/Th2/Th17 analysis, the cells were stimulated for 4 h with 25 ng/mL PMA (Sigma‐Aldrich), 1 mg/mL Ionomycin (Sigma‐Aldrich) and 0.66 μL/mL Golgistop (Sigma‐Aldrich) and cultured at 37°C in 5% CO₂. The cells were harvested and stained with rat anti‐mouse CD3‐PerCP‐Cy™5.5 (BD Pharmingen, San Diego, USA) and rat anti‐mouse CD4‐FITC (eBioscience, San Diego, USA). After being fixed and permeabilized with FIX&PERM Kit (MultiSciences, Hangzhou, China), the cells were intracellularly stained with rat anti‐mouse IFN‐γ‐PE (eBioscience), rat anti‐mouse IL‐4‐PE (BD Pharmingen) and rat anti‐mouse IL‐17A‐PE (BD Pharmingen).
For Tregs analysis, single‐cell suspension was stained with rat anti‐mouse CD4‐FITC (eBioscience) and rat anti‐mouse CD25‐APC (BD Pharmingen). After being washed, fixed and permeabilized with Fixation/Permeabilization Diluent (eBioscience), then the rat anti‐mouse Fc block (BD Pharmingen) was added into cell suspension and incubated for 15 minutes at 4°C. The cells were intracellularly stained with rat anti‐mouse Foxp3‐PE (BD Pharmingen).