Ca2 and mg2 free pbs

Twenty-four hour post-instillation, the rats were euthanized via an intraperitoneal injection of tiletamine-zolazepam (Zoletil®, 50 mg/kg) and xylazine (5 mg/kg). The trachea was cannulated with a blunt 14 gauge needle, and the lungs were lavaged in situ four times with cold sterile Ca²⁺- and Mg²⁺-free PBS (Life Technologies) at a volume of 8 mL. The first lavage was kept for analysis of LDH, total protein, and pro-inflammatory cytokines. Cell pellets from four lavages were pooled for cell counts. The total number of cells in the BALF was quantified by a nucleocounter (Chemometec, Allerod, Denmark), and 4 × 10⁴ cells were attached to glass slides by cytospin at 27 g for 5 min. The cells were then fixed for 5 min with methanol and stained with Diff-Quik (Thermo Fisher Scientific, Waltham, MA, USA) for the differential cell count.

The intratracheal instillation model was selected instead of an inhalation study because the former is an easy and reliable method to identify NP toxicity and compare responses to different particle types [32 (link)]. NP suspensions at 80, 200, and 800 μg/mL were prepared by dispersing NPs in sterile Ca²⁺- and Mg²⁺-free PBS (Life Technologies, Gaithersburg, MD, USA) and sonicated for 10 min using a bath sonicator (Saehan-Sonic, Seoul, Korea) to break up agglomerates. NP suspensions were prepared and sonicated immediately before use as recommend in a previous study [33 (link)]. Six-week-old specific-pathogen-free female rats were purchased from Samtako (Gyeonggi-do, Korea), maintained, and handled according to the policies approved by the Institutional Animal Care and Use Committee of Dong-A University. The instillation (n = 4 per group) was performed according to a previously described method [24 (link)]. NP suspensions were instilled at doses of 40, 100, and 400 μg/rat by instilling 0.5 mL of the suspensions.

Because we hypothesized that NiO NPs can produce eosinophilia via the accumulation of nickel ions in the phagolysosomes of phagocytes, NiCl₂ were instilled into lungs of rats to demonstrate the direct effect of nickel ions. Our previous studies also showed that instillation CoCl₂ and ZnCl₂ produced similar magnitude of eosinophilia as observed in CoO and ZnO at an equal metal concentrations [8 (link), 11 (link)]. Although NiO NPs in the lung might not completely dissolved in the lung within 3 days after instillation, we selected the dose of NiCl₂ at 378.0 μg/rat (171.1 μg Ni/rat), which is equivalent nickel concentration of 200 cm²/rat of NiO NPs to evaluate whether the dissolved nickel from NiO NPs directly induce pulmonary eosinophilia or not. NiCl₂ (Sigma-Aldrich) dissolved in sterile Ca²⁺- and Mg²⁺-free PBS (Life Technologies) was intratracheally instilled into the lungs of rats and then sacrificed at 24 h post-instillation to collect the BALF and serum.

The human erythroleukemic cell line K562(S) from ATCC, was maintained in RPMI-1640 (Roswell Park Memorial Institute) medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (Gibco), 1% 100× penicillin streptomycin and subcultured every 3 days to maintain a cell density of 10 × 105/mL.
Cells were harvested and washed twice by centrifugation with Ca²⁺ and Mg²⁺-free PBS (Gibco) for the agglutination experiments [33 (link)]. The test was conducted with resuspended cells at a concentration of 10 × 10⁷ cells/mL in the same PBS. Briefly, 25 µL of cell suspension was additioned to each well of a 96-well microtiter plate containing digested gliadin (0.75–3 mg/mL) or zein digest (6 mg/mL) concentrations obtained by serial dilution (1:1) with PBS, and then 25 μL PBS was added in each well. The inhibition test was carried out by adding rice hydro-alcoholic extracts (3 mg/mL) instead of 25 μL PBS before the addition of cells to the digested gliadin wells.
The obtained total volume was 100 µL. The cell suspension was incubated at room temperature for 30 min.
Clumped and single cells were counted on the microscope slide after the application of a drop of suspension. Control cells were included, as appropriate. Unless otherwise specified, the agglutination test was repeated twice [33 (link)].

PSCs were passaged at low density in T175 tissue culture flasks and allowed to grow till approximately 50% confluency. Cells were pulsed with 10 μM BrdU (Sigma) for 30 min, Trypsinized, and collected by centrifugation (300g, 5 mins). Cells were washed in Ca²⁺ and Mg²⁺ free PBS (Gibco) and then resuspended in ice-cold 70% ethanol dropwise whilst vortexing and stored at -20°C at least overnight. Cells were incubated sequentially in 1% Triton X-100 PBS (PBST) for 20-30 mins (permeabilization), 2M HCl for 20 mins at room temperature and then washed in PBS-Tween(0.1%). They were then incubated in 0.1M Na₂B₄O₇ for 20 min at RT and washed in PBS-T. Samples were stained with 100 μl α-BrdU (Dako, 0744) diluted in PBS-T for 20 mins, washed twice in PBS-T and then incubated in anti-mouse Alexa fluor 488 secondary antibody (Invitrogen) for 20 mins each. RNA was digested (100 μg/ml RNase 37°C for 15 min) and DNA stained with 50 μg/ml propidium iodide (Sigma-Aldrich) prior to flow cytometry (BD Fortessa) and data analysis (FlowJo).

Male C57BL/6 animals were anaesthetized by i.p. ketamine/xylazine administration and received an intratracheal (i.t.) injection of bleomycin (80 μl, 1 mg/ml) or vehicle (saline) as a negative control. Animals were treated with uridine (80 μl, 24 μg/ml; Sigma Aldrich, Germany) or vehicle at the indicated time points. Mice were sacrificed at different time points (see results section) via i. p. injection of thiopental. BAL was performed with 3 × 1ml of Ca²⁺ and Mg²⁺ free PBS (Gibco, Paisley, UK) supplemented with 0.1 mM sodium EDTA (Sigma Aldrich, Germany), followed by lung resection and storage in OCT freezing medium. BAL cells were counted, differential cell counts were done by FACS analysis, as described previously [12 (link)]. Frozen lung sections were stained with hematoxylin and eosin for histological analysis.