Facs calibar

BAL and lung tissue leukocyte suspensions were stained with anti-mouse CD3, anti-mouse CD4, anti-mouse-TCRδ, anti-mouse Vγ4, anti-mouse IL-17A (BD Pharmingen, Oxford, UK), anti-mouse T1/ST2 (Morwell Diagnostics, Zurich, Switzerland), α-GalCer (Axxora Biochemicals, Farmingdale, NY, USA) loaded CD1d tetramers (ProImmune Ltd., Oxford, UK) or relevant isotype controls. Flow cytometric analysis was performed using a FACSCalibar™ (Becton Dickenson, Oxford, UK) using CellQuest software.

Propidium iodide (PI) and Annexin V-FITC-flow cytometry assay (BD Pharmingen, CA) was used to detect the apoptosis in the cells. Cells were treated with 5-fluorouracil (5-FU) for 18–24 h and harvested in PBS. Then the cells were re-suspended in binding buffer (BD Pharmingen, CA), and stained with FITC-conjugated annexin V and PI (Kaijishengwu, China, KGA107). After staining, the cells were analysed by flow cytometry (FACS Calibar; Becton-Dickinson) using Cell Quest software [21 (link)].

For this experiment, cells were cultured in 6 well plates. Control (untreated) and treated cells with Pb(NO₃)₂ were harvested from 6 well plates after 24 h of exposure. Harvested cells were washed twice with phosphate-buffered saline (PBS) and 5 × 10⁶ cells/mL per sample were fixed with 70% methanol on ice for 10 min. After cells fixation, cell pellet was then suspended in 0.5 mL PBS containing propidium iodide (50 μg/mL) and DNase-free RNase (100 μg/mL) for 15 min. The relative number of cells in the different phases was assessed by flow cytometry (FACS Calibar; Becton-Dickinson) using CellQuest software, and the percentages of cells calculated in G0/G1, S and G2/M phases of the cell cycle.

Apoptotic or necrotic cell levels were determined by flow cytometry after double staining with Annexin V-FITC and propidium iodide using an assay kit from BD Pharmingen as described previously [17 (link)]. Briefly, 2 mL of cells (1 × 10⁶ cells/mL) were added to each well of 12 plates and treated with 5, 10, 20, 40 and 80 mg/mL of D-glucose for 2 h. Control well plates were also made without D-glucose. After 2 h of incubation, 1 × 10⁶ cells/mL were counted and washed in PBS, re-suspended in binding buffer (10 mm Hepes/NaOH pH 7·4, 140 mm NaCl, 2·5 mm CaCl₂), and stained with FITC-conjugated annexin V (Pharmingen, Becton Dickinson Co., San Diego, CA, USA). After staining, the cells were incubated for 15 min in the dark at room temperature. Cells were re-washed with binding buffer and analysed by flow cytometry (FACS Calibar; Becton-Dickinson) using Cell Quest software.

Fresh leaves were used for ploidy identification using a flow cytometer (FACSCalibar; Beckton Dickinson Co., Franklin lakes, NJ, United States) following the manufacturer’s instructions. About 0.5 cm² of leaf disk was dipped in 400 μL extracting buffer [1% beta-mercaptoethanol, 0.05% Triton X-100, 20 μg mL^–1 RNase A, 15 mM Tris–HCl (pH 8.0), 2 mM Na₂EDTA, 20 mM NaCl, and 80 mM KCl], ground into small particles (Zhang et al., 2011 (link)), and filtrated through a 500-μm mesh sieve. The filtrate was stained with 20 μg mL^–1 propidium iodide (Sigma, Louis, Missouri, United States) and incubated in the dark for 15 min at room temperature. After staining, the nuclei were collected by filtering through a 25-μm nylon mesh. Flow cytometry was performed using the flow cytometer. Diploid “Hanfu” apple (M. domestica, 2n = 2x = 34) was used as a control and internal reference (Ma et al., 2016 (link)). All chemicals were purchased from the TransGen Biotech Co., Ltd. (Beijing, China) unless otherwise indicated.

Propidium iodide (PI) and Annexin V-FITC-flow cytometry assay (BD Pharmingen, CA) was used to detect the apoptosis in the cells. Cells were stained with FITC-conjugated annexin V and PI (KeyGEN BioTECH, China, KGA107) according to the manufacturer’s instructions. After staining, the cells were analyzed by flow cytometry (FACS Calibar; Becton-Dickinson) using Cell Quest software.
TUNEL assay was processed using the in situ cell death detection kit (KeyGEN BioTECH, China, KGA7071). Hepatic tissues were stained according to the manufacturer’s instructions. The apoptosis index was captured using a fluorescence microscope (Olympus IX73, Japan).