Facs calibre flow cytometer

After the addition of drugs at indicated time points, cells were collected and washed with ice cold PBS and fixed with ice cold 70% ethanol. Cells were treated with 500 µg/ml of RNase A (Sigma) and subsequently stained with 50 µg/ml of propidium iodide (PI, Sigma) for 15 minutes at 37°C. The populations of cells stained by PI were then analysed by FACS calibre flow cytometer (Beckton Dickinson).

MCF-7 cells were seeded at density of 2 × 10⁵ into each well of a 6- well plate. The cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM), supplied with 10% foetal bovine serum, and incubated in a humidified atmosphere containing 5% CO₂ in air for 24 h at 37 °C. The incubation medium was replaced with a fresh one containing the synthesised hybrids 6a and 6i at their IC₅₀ in DMSO (1% v/v). The cell plates were incubated for 24 h. The cells were washed twice with cold phosphate buffered saline (PBS) then fixed with 70% ice-cold ethanol. Cells washed with PBS at 37 °C for 30 min and recovered by centrifugation at 2000 rpm for 5 min. Cells were stained using DNA fluorochrome propidium iodide (PI). The plates were incubated at room temperature for 20 min in a dark place. Then the cells were investigated with a FACS Calibre flow cytometer (Becton Dickinson, Heidelberg, Germany)⁴⁸ .

Caco-2 IECs were harvested and resuspended to a density of 2 × 10⁶ cellsml⁻¹; 100 μL aliquots were washed twice in sterile DPBS and incubated with 2% w/v BSA in Ca²⁺/Mg²⁺ free PBS for 30 min on ice to enable blocking of non-specific binding of antibodies. Cells were washed in 1% w/v BSA/PBS and incubated with the appropriate PE fluorochrome-conjugated antibody (1/200 dilution anti-TLR2 Clone TL2.1, anti-TLR4 Clone HTA125, and appropriate isotype-matched control antibodies)(eBiosciences, UK) for 30 min at 4 °C in the dark. Cells were washed twice in 1% w/v BSA/PBS to remove unbound antibody and resuspended in 500 μL 1% w/v BSA/PBS and kept on ice in the dark, until ready for data acquisition and analysis. TLR2 and TLR4 staining was detected by analysing the cell samples in a FACS Calibre Flow Cytometer (Becton Dickinson, San Jose, CA, USA) and analysed by BD FACS Diva Software v6.0. Positive staining for these membrane receptors is expressed as Net mean fluorescence intensity (Net MFI) obtained by subtracting isotype control sample MFI from positive test MFI for at least 3 replicate samples live gated by FSC/SSC and positivity determined by setting 5% confidence interval gating on live cells.

To evaluate HR repair of DNA-DSBs, Hela cells containing the DR-GFP construct were transfected with the I-SceI expression vector. Transient expression of I-SceI endonuclease generates a DSB at the integrated green fluorescent protein (GFP) gene sequences and stimulates HR. 24 h later, the cells were transfected with Aurora A siRNA, Bora siRNA, or various Bora expression plasmids. GFP signal was assayed 3 days post-transfection on a FACSCalibre flow cytometer (BD, Biosciences).

Apoptosis induction was assayed by staining K562 cells with Annexin-V (fluorescein isothiocyanate) and the counterstaining PI using the Annexin-V-FITC/PI apoptosis detection kit (BD Biosciences, San Diego, CA) according to the manufacturer’s instructions. Cells were exposed to the tested compound for 48 h. After that, the cells were collected by trypsinisation and 0.5 × 10⁶ cells and washed twice with phosphate-buffered saline (PBS) followed by staining with 5 µl Annexin-V-FITC and 5 µl PI in 1 × binding buffer for 15 min at room temperature in the dark. FACS Calibre flow cytometer (BD Biosciences, San Diego, CA)²⁶ (link)^{,36 ,37} was used for analyses.