T25 flask

Cells were seeded in T25 flask (Greiner bio-one, Frickenhausen, Germany) for 24 h. After reaching ~80–90% confluence, cellular uptake assay was performed. Cells were treated with fresh media (100 μL) containing the QCT-loaded SLNs (equivalent to 5 μg/mL QCT). Following incubation for 4 h, cells were washed two times and harvested using trypsin/EDTA. Then, pellets were collected by the cooling centrifuge. QCT was extracted by ethanol and quantified by HPLC. QCT was analysed by Waters (Milford, MA, USA) 2690 Alliance HPLC system equipped with reversed phase stainless steel column (250 mm × 4.6 mm), packed with 5 μm particles (Inertsil ODS-3V, Dikma Co., Foothill Ranch, CA, USA) and a Waters 996 photodiode array detector. The mobile phase was composed of 0.1% phosphoric acid in water and acetonitrile (60:40, v/v). The flow rate was adjusted to 1 mL/min, the wavelength of UV detector was kept at 254 nm while samples (10 μL) were injected into the column. All procedures were performed at room temperature. The method showed good linearity (R² = 0.994) over the tested concentration range (50–250 ng/mL).

8- to 12-week-old C57/black 6 mice were killed using CO2, and their femora and tibiae were removed and cleaned of skin and muscle. According to the method of Dobson [25] (link), the proximal and distal ends of the murine femora and tibiae were removed, the bones were inserted into Eppendorf tubes, and bone marrow was obtained after centrifugation (10 min, 250×g). Bone marrow cells were then collected in medium (RPMI 1640, 10% foetal calf serum [FCS], 1% 1 M HEPES, 0.5% gentamycin, and 0.1% mercaptoethanol; all purchased from Gibco, Invitrogen, Germany). To obtain adherent cells, 5.4×107 cells per T25 flask (Greiner, BIO, Germany) were plated and cultured at 5% CO2 and 37°C. After 72 h, the supernatants, and therefore all suspended cells, were discarded, and the medium was replaced. On day 7 (cells nearly reached confluence), the cells were either used for coculture with CIKs or were harvested for flow cytometry.
The medium in some of the flasks was removed after 6 days and replaced with 3.5 ml of fresh medium supplemented with DiD staining solution (5 µl DiD/ml; Vybrant staining solution, Invitrogen). After incubation with the staining solution for 30 min, the supernatant was discarded, and the cells were washed with phosphate-buffered saline (PBS). Finally, fresh medium was added and DiD-labelled cells were placed in the incubator until use the following day.

APO-DIRECT^™ Apoptosis Kit (Novus Biologicals, Littleton, CO) was used according to the manufacturer's instructions. MCF7 cells were cultured overnight at a seeding density of 3×10⁵ cells/T25 flask (Greiner Bio-One GmbH, Germany) with 15 mg/ml and 25 mg/ml concentrations of MEAD for 48 h. Cells were trypsinized, washed with DPBS, fixed with 1% paraformaldehyde (Sigma, St. Louis, MO) for 60 min at 4°C, washed twice with DPBS and then resuspended in ice-cold 70% ethanol and stored overnight at -20°C. They were then centrifuged and washed twice with wash buffer and incubated overnight with 50 μl of DNA labeling solution at RT in the dark. The reaction was stopped by washing twice with rinse buffer and the cells were incubated with PI/RNase solution in the dark at RT for 30 min before being analyzed using flow cytometer (FACS Aria III, BD Biosciences, San Jose, CA).

Both cell lines were seeded at a density of 1 × 10⁶ cells per T25 flask (Greiner), and 2 days after seeding [195mPt]cisplatin (Pt-0011, Pt-0016) was added in three concentrations (5 µM, 20 µM and 75 µM). To assess uptake and retention of [195mPt]cisplatin we performed, the experiment under three conditions: (A) 4 h exposure to [195m]cisplatin and after which cells were harvested, (B) 4 h exposure to [195mPt]cisplatin after which the cisplatin-containing medium was removed, cells were rinsed with PBS and cisplatin-free DMEM was added and left for 24 h and (C) 24 h incubation with [195mPt]cisplatin after which cells were harvested. Cells were analyzed under the microscope for viability. For further analysis of all different fractions the cell suspension was centrifuged for 5 min at 500xg, the cell fraction was rinsed with PBS and collected. Cells were lysed and homogenized, and both RNA and DNA were isolated using the AllPrep DNA/RNA mini kit (Qiagen). The radioactivity of all fractions was determined using a gammacounter (LKB-Wallac, 1282 CompuGamma; Pharmacia, Woerden), and corrected for decay.

Human neonatal foreskin fibroblast line HF043 (Dundee CELL products) was cultured in DMEM (Sigma) supplemented with 10% fetal calf serum (Gibco) in the absence of any added antibiotics, at 37°C in a humidified incubator with 5% CO₂. Cells were monitored microscopically using an EVOS digital microscope (Life Technologies) and harvested when ∼80% confluent using TrypleExpress (Invitrogen). Following resuspension in DMEM with FCS, 20 μl of the cell suspension was counted and cell diameters measured using a Cellometer T4 (Nexelcom). Cells were seeded at 2×10⁵ per T25 flask (Greiner), or at 1×10⁴ in 24 well plates (Greiner). Population doublings (PD) were calculated as:
$\mathit{\text{PD}}=\frac{{\log }_{10}(\mathit{\text{total}} \mathit{\text{cells}} \mathit{\text{harvested}} / \mathit{\text{total}} \mathit{\text{cells}} \mathit{\text{seeded}})}{{\log }_{10}2}$
Cumulative population doublings (CPD) were calculated as the sum of PD values.
AZD8055 (Selleckchem) was reconstituted to 1mM in DMSO and stored in aliquots at −20°C protected from light. Prior to drug treatment, medium was removed, cells washed with PBS, then fresh medium supplemented with drug was added. Total volume of drug or DMSO added to the culture medium never exceeded 1:10,000 v/v. Doses of 35 nM and 70 nM were chosen as effective but non-toxic based on our preliminary studies (not shown).

AML12 cells were purchased from ATCC (CRL-2254) and cultured in 1:1 DMEM and Ham’s F12 medium (Thermo Fisher Scientific), supplemented with 10% FBS, 1% penicillin/streptomycin (100 U/ml and 100 μg/ml, respectively), 1% insulin-transferrin-selenium (ITS; 10 mg/l, 5.5 mg/l, and 6.7 μg/l, respectively), and dexamethasone (100 μmol/l) at 37°C in 5% CO₂. Cells were removed from liquid nitrogen, thawed rapidly, and initially cultured in a T25 flask (catalog 690175, Greiner). Medium was changed every 2 days, and upon reaching confluence, cells were subcultured in T75 flasks (catalog 7340290, VWR) at a 1:3 ratio. Cells were plated at a density of 50,000 cells/well in collagen 1–coated 12-well plates (catalog 7340295, VWR) and grown to confluence in maintenance medium. Cells were then supplemented with low glucose (7.60 mmol/l) with unlabeled l-glutamate (4 mmol/l) and dialyzed FBS (Thermo Fisher Scientific) for 2 days. After switching medium, cells were serum starved for 20 hours in low-glucose medium supplemented with unlabeled l-glutamate (4 mmol/l).