Coulter epics xl mcl

Flow cytometric analyses were performed using a Coulter EPICS XL-MCL (Beckman Coulter, Pasadena, CA), a BD LSR Fortessa (Becton Dickinson, Erembodegem, Belgium) or a BD FACSCanto II (Becton Dickinson). Collected data were exported to the FACSDiva 6.0 software (BD Biosciences) to select subpopulations according to their forward and scatter factors.

According to the manufacturer’s instructions, cardiomyocyte apoptosis was determined by flow cytometry using the Annexin V-FITC Apoptosis Detection Kit (Beyotime, Shanghai, China). In brief, cardiomyocytes were stained with a mixed dye solution of Annexin V-FITC (an indicator of apoptosis) 5 μL (20 μg/L) and propidium iodide (PI) 10 μL (50 μg/L) at 4°C for 30 min incubation. Analysis by flow cytometry (COULTER EPICS XL-MCL; Beckman Coulter, Brea, CA, United States) was performed within 30 min (excitation and emission wavelengths of 485 and 525 nm, respectively).

Sternum fine-needle (18G) BM aspiration (3–5 ml) was performed by an expert haematologist in the surgical theatre, once the patient was under general anaesthesia and before the sternotomy. Fresh BM smears were used for direct cell count (only in COPD patients). A 50 μl BM sample was diluted 1:4 in 1× PBS (pH 7,4; PAA) and analyzed using CELL-DYN Sapphire (Abbot, USA) to determine cell types distribution. HSC and EPC were isolated from fresh BM aspirates using a RosetteSep Kit (STEMCELL Technologies Inc., Canada) following manufacturer’s instructions to obtain a more representative sample. Cells were quantified by flow cytometry on a FACScan (Coulter Epics XL-MCL; Beckmann Coulter) by the presence of antigens CD34 (BD Pharmingen, NJ, USA), KDR (R&D Systems, Minneapolis, USA), CD133 (Miltenyi Biotec, Germany), c-kit, Ki67 (Abcam, Cambridge, UK). Gates were set to detect CD34+ cells and to evaluate co-expressions of CD34+ cells with CD 133, KDR, c-kit and Ki67. At least 20.000 events were acquired for each sample. Following previous publications [2 (link)–4 ], EPCs were also identified by FACS as CD34+ CD133+ KDR+ cells. The remaining BM sample were centrifuged at 1800 rpm/5 min/4 °C to separate cells from plasma and the supernatant was stored (100 μl aliquots) at − 80 °C until cytokine analysis.

The viability of tendon cells after applied cyclic strain was assessed with propidium iodide staining, as previously described [16] (link). The strained and unstrained tendon cells were trypsinized and centrifuged along with their supernatant in individual tubes. The cell pellets were washed in PBS and fixed with ice cold 70% ethanol for 1 hour. Then the fixed cells were stained with propidium iodide staining solution [1 ml PBS (Ca⁺², Mg⁺² free, 0.1% glucose), 10 microliters Rnase A 100 mg/ml and 5 microliters propidium iodide 10 mg/ml] and incubated for minimum 30 minutes. The DNA content in suspended cells was analyzed using the FL3 channel on a flow cytometer, Coulter Epics XL- MCL (Beckman Coulter Inc, USA), and cell death was calculated based on cell populations in the sub G1 phase of the cell cycle.

To determine the effect of CASE on cell cycle progression, LNCaP cells (1×10⁶) were plated in 75 cm² flasks and allowed to attach for 36 h. The media was replaced with fresh serum-complete medium containing CASE and cells were treated for 12 h. Cells were harvested by trypsinization, washed with phosphate buffered saline (PBS), and fixed with 70% methanol. Cells were then treated with RNase (500 μg/mL) and stained with propidium iodide (40 μg/mL) for 30 min at 37°C. The cells were analyzed by flow cytometry using a Coulter Epics XL-MCL (Beckman Coulter, USA).