Cd3 fitc

Changes in T cell sub-populations were quantitated by flow cytometric analysis as described
[18 (link),19 (link)]. CD8-PE-Cy5, CD16-PE, CD25-APC, CD45RA-FITC, CD45RO-PE, CD56-APC, CD127-PE, and CD152-PE-Cy5 were purchased from BD Biosciences (San Jose, CA). CD3-FITC and CD4-APC were purchased from Miltenyi Biotec (Auburn, CA).

All animals were cared for in a specific pathogen-free room and treated following the animal care protocol approved by an animal committee. Mouse LLC1 (1 × 10⁶) cells were subcutaneously injected into 8-week-old -C57BL/6 mice. After one week of inoculation, the mice were intraperitoneally administered melatonin (30 mg/kg, three times per week) for 4 weeks. The tumor volume and mice body weights were monitored every 2 days during the experiments. Tumor volume was calculated using the following equation: length × (width)2 × 0.5. To analyze tumor-infiltrating lymphocytes (TILs), freshly isolated tumor tissue was cut into small pieces and disassociated by the gentleMACS tumor dissociation kit (Miltenyi Biotec). The suspension was further treated with a RBC lysis buffer to remove red blood cells. Approximate 1 × 10⁶ isolated cells were incubated with fluorophore-conjugated antibodies including CD45-APC, CD11b-PE, CD3-FITC, CD8-APC-Vio770, Ly6G-FITC, CD4-PE-Vio770, and F4/80-APC (Miltenyi Biotec) and analyzed by the BD FACSVia flow cytometer (BD Biosciences) [43 (link)].

PBMC samples were seeded (1 × 10⁴ cells/well) in complete medium with or without the indicated concentrations of compounds. After 72 h incubation, cells were washed with PBS and stained with CD45RA PE (BD-Biosciences), CD62L APC (Miltenyi Biotec), CD3 FITC (Miltenyi Biotec) at room temperature for 20 min. Data collection was done on FACSVerse and dead cells were excluded from the analysis, based on the use of PI (Sigma-Aldrich).
For the evaluation of hENT2 protein expression, cell lines or AML primary cells were incubated with a SLC29A2 polyclonal antibody (Thermo fisher Scientific) using a dilution of 1:25 for 20 min at room temperature after fixation and permeabilization. The secondary antibody (Cell signaling Technology USA, Danvers, MA, USA) was used with a dilution of 1:200 for 20 min. Results were expressed as ΔGmean, i.e., the net mean fluorescence intensity (MFI) difference between the cells stained with primary and secondary antibodies and cells stained with the secondary antibody only, in order to eliminate the background positivity. All data were processed with Kaluza software version 1.2 (Beckman Coulter).

Peripheral blood mononuclear cells (PBMCs) were isolated from 20 to 25 ml of peripheral blood samples via Ficoll density gradient centrifugation (Amersham Biosciences, Uppsala, Sweden). According to the recommendation of the international working group of the experts in flow cytometry [7 (link)], CD3, CD4, CD25, CD127, and FoxP3 markers were used to analyze human Treg cells using a gating strategy illustrated in Supplementary Figure 1. Specifically, CD3-FITC, CD45-PerCP, CD127-APC (Miltenyi Biotec, Bergisch Gladbach, Germany), CD4-Alexa Fluor® 700, (EXBIO a.s., Prague, Czech Republic), Foxp3-PE, and CD25-Brilliant Violet (BioLegend, San Diego, USA) antibodies (Abs) were used. For intracellular staining, we used Foxp3-Fix/Perm and Foxp3-PErm Buffers (BioLegend, San Diego, USA). The flow cytometric data were collected on a LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using Summit 4.3 software (Dako, Glostrup, Denmark).

K562-luc2 cells were thawed from cryopreservation 48h before tumor injections and maintained at 37°C, 5% CO₂ in Iscove’s DMEM supplemented with 10% FBS and 8µg/mL blasticidin for 2 days. On the day of tumor injections, K562-luc2 cells were removed from culture, washed three times with sterile PBS, and resuspended at a concentration of 1x10⁶ cells/200µL in sterile saline. Cryopreserved PBMCs were thawed in a 37°C water bath for two minutes or until a small ball of ice remained in solution. PBMCs were resuspended in 5mL RPMI + 10% FBS. A small aliquot (50 µL) was taken, and cells were labeled with monoclonal antibodies (CD8-VioBlue, CD14-VioGreen, CD3-FITC, CD4-PE, CD20-PerCP, CD45-APC, CD56-APC-Vio770 (Miltenyi Biotec) for surface staining prior to injection. The remaining cells were supplemented with IL-15 (0.1mg/mL) and incubated for 1 hour at 37°C, 5% CO₂ to improve activation and recovery of NK cells functions. After incubation, PBMCs were washed three times with PBS to remove all RPMI media and then resuspended at a concentration of 10x10⁶ PBMCs/200µL sterile filtered saline for injections.