Facsvantage

For detection of the cell differentiation antigen, CD11b, ATRA (Sigma-Aldrich; Merck Millipore) was used to induce cell differentiation at a concentration of 1 nM for 3 days. The cells (1×10⁶/group) were washed twice with PBS and incubated with phycoerythrin (PE)-conjugated CD11b antibody (cat. no. 12-0113-42; eBioscience, Inc., San Diego, CA, USA) at 4°C for 30 min in the dark. The cells were then analyzed using flow cytometry (BD FACS Vantage; BD Biosciences, San Jose, CA, USA) and CellQuest Pro software version 5.1 (BD Pharmingen, San Diego, CA, USA).

The U251MG cells were plated in six-well plates. When cell confluence reached 80%, the liposomes and free curcumin were respectively added into the media at a final concentration of 20 μM. After another 48 h incubation, the cells were collected with 0.25% trypsin/EDTA and centrifuged at 1,000 rpm for 5 min, and then fixed in 70% ethanol for 24 h. Afterwards, cell cycle (propidium cell cycle assay kit; Beyotime Inst. Biotech., Shanghai, People’s Republic of China) was measured using a flow cytometer (BD FACSVantage; San Jose, CA, USA). Moreover, after the cells were treated with RCL (final concentration, 20 μM), cell apoptosis (Annexin V-PE apoptosis detection kit; Beyotime Inst. Biotech.) was measured by flow cytometry.

CD8CD44^lo OT-1 T cells (5 × 10⁶ cells) were loaded with 1.8 µM Indo-1 (ThermoFisher Scientific, Cat. No. 1223) in Indo buffer (HBSS with calcium and 0.5% BSA) at 37 °C for 30 min. The cells were washed twice with the buffer and let stand for 40 min at 4 °C with a gentle shaking at 20 min, followed by washing twice in Indo buffer. The cells resuspended in the buffer (0.5 mL) were heated to 37 °C for 2 min, added with DCs (5 × 10⁶ cells in 0.5 mL), and spun using a minifuge for 20 s to have the T cells contact with DCs, without IL-2. Various times later (1 to 8 min), the cell mixture was resuspended by a short vortexing and applied to a flow cytometer (BD FACSVantage, BD Bioscience). The 405/510 nm emission signal ratio for Indo-1 (Indo-1 ratio) was used to analyze the intracellular Ca²⁺ flux. The peak of median of the top 60% Indo-1 ratios for each of the samples incubated for different times was plotted against the duration of DC-T cell contact to obtain intracellular Ca²⁺ concentration changes induced by the contact with DCs. In some experiments, DCs loaded with ovalbumin peptide (SIINFEKL, American Peptide Company/Bachem) were used to induce TCR stimulation-mediated Ca²⁺ flux as a control.

For mice, the following antibodies were used: Alexa Fluor 647 anti-CD144 (eBioBV13), PE anti-CD45 (30-F11), PE-Cy7 anti-CD31 (390), APC- eFluor 780 anti-c-kit (2B8), PE-anti-ESAM (1G8/ESAM), APC anti-CD19 (1D3), PE-Cy7 anti-B220 (RA3-6B2), PE-Cy anti-Thy1.2 (53-2.1), APC-C7 anti-CD25 (PC61), PerCP-Cy5.5 anti-Gr-1 (RB6-8C5), PE anti-CD3ɛ (145-2C11), APC-Cy7 anti-Mac-1 (M1/70), FITC anti-CD45.2 (104), PE-Cy7 anti-CD45.1 (A20), PerCP-Cy5.5 anti-Sca1 (D7), eFluor 450 lineage-specific cocktail [(anti-TER119 (TER119), anti-Gr-1 (RB6-8C5), anti-B220 (RA3-6B2), anti- CD3 (17A2), and anti-Mac-1 (M1/70)], APC anti-CD34 (MEC14.7), PE anti-Flt3 (A2F10), PE anti-IFNgr1 (2E2, Abcam), APC anti-CD48 (HM48-1), and PE-Cy7 anti-CD150 (TC15-12F12.2). DAPI was used to exclude dead cells. Cells were sorted on a BD FACSvantage at low speed and low pressure (North et al. 2002 (link)) and analyzed on a BD LSR II flow cytometer; data were analyzed with FlowJo (Tree Star).
For zebrafish, transgenic embryos (five per sample with four or more replicates) were incubated in 0.5 mg/mL Liberase (Roche) solution with gentle agitation for 90 min at 37°C and then dissociated, filtered, and washed with PBS. Dead cells were excluded by 5 nM SYTOX red (Life Technologies) prior to analysis using a BD FACS Canto II or Beckman Coulter Gallios. Cell sorting was performed using a Beckman Coulter MoFlo XCP.