Annexin 5 allophycocyanin apc

Cell apoptosis was conducted by staining with Annexin V-Allophycocyanin (APC) and 7-amino-actinomycin (7-AAD) (BD Biosciences, Erembodegem, Belgium) for 15 min at room temperature in the dark, followed by flow cytometric analysis within 1 h (BD Biosciences). Cell apoptosis was analyzed using the WinMDI 2.9 software (BD Biosciences).

LPS was purchased from Sigma Aldrich (St Louis, MO, USA) and originated from S. Typhimurium (LPS1) and E. Coli (LPS2), respectively. All stimulations were performed for a total of 6 h except for rhS100A9 (20 h). IL-1β and HMGB1 was from R&D Systems. Recombinant human S100A9 (rhS100A9) was a gift from Active Biotech AB and a detailed description on endotoxin-free S100A9 generation and purification has been published previously [15 (link)] and was used in the presence of calcium and zinc (Ca²⁺ ≥200 μM; 10 μM ZnCl₂ [34 , 35 (link)]). Supernatants from stimulated or siRNA transfected cells were harvested and analyzed using human inflammatory cytokine cytometric bead array (CBA; BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions or using IL-6 and IL-8 Quantikine ELISA (R&D Systems, Minneapolis, MN, USA). Annexin V-allophycocyanin (APC) and propium iodide (PI) staining was performed according to the manufacturer’s instructions (BD Biosciences). The cycloheximide (CHX) experiments (Sigma Aldrich) where performed by adding 10 μg/ml CHX, with or without 100 ng/ml LPS for 6 h.

Cells used for cell cycle analysis were plated 24 h prior to transfection with miRNAs in 24-well plate using Lipofectamine 2000 per manufacturer's instructions. Cells were trypsinized and then collected by centrifugation at 2,000 rpm for 6 min. Cell pellets were rinsed once with phosphate-buffered saline (PBS) and fixed in 70% (v/v) ice-cold ethanol for at least 16 h at -20°C. Cells were resuspended and stained with 50 μg/mL propidium iodide (BD Biosciences, Erembodegem, Belgium) containing RNase A for 30 min at 37°C in the dark. Apoptosis was determined by dual staining with Annexin V-Allophycocyanin (APC) and 7-Aminoactinomycin D (7-AAD) (BD Biosciences, Erembodegem, Belgium). Briefly, cells were collected 48 h after transfected with pre-miR-139 or control, and stained with Annexin V-APC and 7-AAD according to the manufacturer’s instruction. The combination of Annexin V-APC and 7-AAD staining distinguished early apoptotic cells (Annexin V+, 7-AAD-) and late apoptotic cells (Annexin V+, 7-AAD+). The cells were sorted by FACS-Calibur System (BD Biosciences) and cell-cycle profiles and apoptosis were analyzed by ModFit 3.0 software (BD Biosciences). The assay was carried out in triplicate for three times.

To analyse the effects of miR-134 on apoptosis induction, cells were transfected with miR-134-mimic or NC-mimic and, 24 h later, transfected cells were treated with 15 μM cisplatin for a further 24 h. CM was then collected and cells were trypsinised and pelleted. CM was used to neutralise trypsin and to collect any apoptotic cells which may be present in the CM. Cell pellets were re-suspended in 2 ml 1X binding buffer (BB) (0.1 M HEPES, 1.4 M NaCl, 25 mM CaCl₂, pH 7.4), centrifuged at 200 g and supernatant discarded and resuspended in 30 μl of BB solution. 20 μl of cell suspension, 5 μl of Annexin-V-allophycocyanin (APC) (BD Biosciences) and 5 μl of propidium iodide (PI) staining solution (BD Biosciences) were mixed. 70 μl of 1X BB was added and incubated at room temperature in the dark for 15 mins. 400 μl of 1X BB solution was subsequently added and mixed by pipetting. Levels of apoptosis was analysed on 2 × 10⁴ cells using a BD Accuri™-C6 flow cytometer.

SK-HEP1, HUVEC, or HHSECs were seeded at a density of 4–6 x 10⁴ cells/well in a 24-well plate. The cells were then treated with varying concentrations of either Bu (200 μM, 300 μM, and 500 μM) or Treo (3 μM, 10 μM, and 30 μM) for 48h [doses decided based on previous publications] [35 (link), 69 (link)]. Apoptosis was determined by staining the cells with Annexin V-allophycocyanin (APC) and 7AAD (BD Pharmingen, San Diego, CA) per the manufacturer’s instructions. The cells were acquired using the Accuri C6 cytometer (BD Biosciences, Franklin Lakes, NJ) and analyzed using BD Accuri C6 software (version 1.0.264.21).