Heat inactivated fbs

Cryopreserved PBMC were quickly thawed, washed and resuspended in RPMI 1640 medium (Sigma, Dorset, UK) supplemented with penicillin (100 U/mL), streptomycin (100 mg/mL), L-glutamine (2 mM) (Invitrogen, Paisley, UK) and 10% heat-inactivated FBS (Biosera, Uckfield, UK). PBMC were set up for ex-vivo ELISPOT at 8 × 10⁶/mL (50 mL/well of capture antibody-coated Millipore plate) for 20 h incubation, as optimized and described previously 6 (link),9 (link). ELISPOT kits were purchased from Mabtech (Nacka, Sweden) and manufacturer's instructions followed for plate development, with modifications as previously described 9 (link). The CMV and EBV viral antigenic stimuli used, were pools of known CD8 epitope peptides (10 µg/mL) for CD8 responses 10 (link) and pools of known CD4 epitope peptides (10 µg/mL) plus viral lysates for CD4 responses. Medium-only and phaetohaemagglutinin (PHA, 5 µg/mL) controls were used in all assays. Measurement of polyclonal secretion of IL-5, IL-17 and IL-2 was carried out using PHA as the stimulus, and ELISPOT reagents from Mabtech (IL-5), eBioscience (IL-17), or R&D Sytems (IL-2), again following manufacturer's instructions. Results are expressed as spot-forming cells/10⁶ PBMC (net antigen-stimulated spots minus medium).

A375 human skin melanoma cells and HeLa
human cervix adenocarcinoma
cells were purchased from ECACC (UK). HCT166 and MRC5pd30 were obtained
from ATCC, Manassas, VA, USA. A375, HeLa, HCT116, and MRC5 cells were
cultured in the DMEM growth medium (high glucose, 4.5 g L^–1, Biosera) supplemented with gentamycin (50 mg mL^–1) and 10% heat-inactivated FBS (Biosera); media for the MRC5 cells
were further enriched by 1% nonessential amino acids (Sigma-Aldrich,
Prague, Czech Republic). A human telomerase reverse transcriptase
(hTERT)-immortalized EP156T prostatic epithelial cell line was purchased
from the American Type Culture Collection (CRL3289, ATCC, Manassas,
VA, USA).
For the biological experiments, the stock solutions
of Ir complexes were prepared in DMSO and further diluted to the EBSS
or DMEM medium. The final concentration of DMSO in biological experiments
did not exceed 1%.

Tibias and femurs from C57BL/6 mice were removed using sterile techniques and the BM cells were flushed out of the cavity. The red blood cells (RBCs) were lysed with RBC lysis buffer (Tiangen Biotech, Beijing, China). To obtain BM-derived MDSCs, 2.5 × 10⁶ BM cells were plated into dishes with 100 mm diameter in 10 ml of medium and induced by GM-CSF (40 ng/ml) and IL-6 (40 ng/ml) cytokines (R&D Systems, Inc., MN, USA).^{42 (link)} The cells were maintained at 37 °C in 5% CO₂-humidified atmosphere for 4 days in the culture medium with RPMI 1640 (Biosera, Boussens, France), 150 U/ml streptomycin, 200 U/ml penicillin and 10% heat-inactivated FBS (Biosera). After 4-day induction, MDSCs were sorted on a FACS Aria II (Becton Dickinson, San Diego, CA, USA) by using mAb CD11b (clone: M1/70), Ly-6G (clone: RB6-8C5) and Ly-6C (clone: HK1.4) (eBioscience, San Jose, CA, USA). Flow cytometry verified that all of the isolated MDSCs yielded a pure population of more than 90%. The sorted MDSCs were cultured in the same medium as described above.