Dulbecco s phosphate buffered saline dpbs

Cells were cultured into flat-bottom 96-well plates (Nunc), with 1500 cells per well in full growth medium overnight. Next, the cells were treated with cisplatin, topotecan (a topoisomerase inhibitor used to treat ovarian, lung, and many other cancer types), and Q-VD for 72 h, as described above. After treatment, the cells were stained with the LIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells (Thermo Fisher Scientific) by diluting 2 μM of calcein-AM and 4 μM of ethidium homodimer-1 (EthD-1) in Dulbecco’s phosphate-buffered saline (DPBS, PanEco, Moscow, Russia).
For the quantitative evaluation of viable and dead cell numbers, VarioScan Flash microplate reader was used. The Calcein-AM signal from viable cells was detected 15 min after dye addition using excitation and emission filters at 485 nm and at 517 nm. The EthD-1 signal from dead cells was detected 45 min after dye addition using excitation and emission filters at 530 nm and at 617 nm. Empty wells were used for background signal estimation; cells incubated with 70% ethanol for 30 min were used as a positive control for EthD-1 staining.

Umbilical cord blood was obtained from healthy full-term pregnant women with gestational age 39–40 weeks with in accordance with the Protocol and Standards of the Stem Cell Bank of Kazan State Medical University. The study was approved by the Institutional Review Board of Kazan State Medical University. Written informed consent was obtained from each subject according to the clinical and experimental research protocol, approved by the Local Ethic Expert Committee of the Kazan State Medical University (number 195, 10 May 2010). Isolation of mononuclear blood cells and generation of adenoviral vectors was performed as described previously [10 (link)]. After purification, UCB-MCs were maintained in RPMI-1640 medium (Sigma, USA) supplemented with 10% FBS (Sigma), penicillin and streptomycin (100 U/ml, 100 μg/ml, respectively, Sigma). Immediately after isolation, UCB-MCs were seeded in 10 cm culture dishes and transduced with the recombinant adenoviruses Ad5-VEGF and Ad5-GDNF, or Ad5-EGFP (enhanced green-fluorescent protein) at an MOI of 10. Cells were incubated for 12–16 h in a humidified atmosphere of 5% CO₂ at 37°C. Prior to injection, the cells were washed with Dulbecco`s Phosphate Buffered Saline (DPBS, Paneco, Russia).

The A. fumigatus strain AfS150 [22 (link)], generated from ATCC 46645 and expressing the dTomato fluorescent protein, was used in this study. The fungus was grown at 37°C on Aspergillus minimal medium supplemented with 1% D-glucose as the carbon source. A fungal suspension was transferred to AMM agar plates and incubated for 3 days at 37°C. Conidia were harvested in 0.01% Tween 20–Dulbecco's Phosphate-Buffered Saline (DPBS) (PanEco, Russia) solution.
Conidia were then fixed overnight with 3% paraformaldehyde (Sigma-Aldrich, USA), washed twice with DPBS, filtered through Steriflip Filter Units (Millipore, Ireland), aliquoted, and stored at 4°C until use. Since the fluorescence of dTomato fluorescent protein was lost after fixation, they were labeled with Alexa Fluor 594 NHS Ester (Thermo Fisher, USA, A20004) for visualization, according to the manufacturer's instructions. Alexa Fluor 594-labeled spores were then filtered through Steriflip Filter Units (Millipore, Ireland), aliquoted, and stored at 4°C until use.