Cyan adp analyser

The chimeric genes coding for the single-chain peptide-β₂-microblobuline-MHC-E trimers were constructed as described previously [47 (link),48 (link)]. Briefly, the DNA fragment coding the tested peptide was inserted downstream of the fragment coding for the signal sequence of HLA-E*01:01 followed by coding sequences for a flexible linker [G₄S]₄, β₂-microglobulin and the desired heavy chain of MHC-E. The Ala at position 84 was mutated to Tyr to accommodate for the flexible linker. The assembled plasmids were then transfected into HEK 293 T cells maintained between 10% and 90% confluency at 37 °C, 5% CO₂ in DMEM (Life Technologies) supplemented with 10% FBS (SeraLabs), and 50 units/ml Penicillin/50 μg/ml Streptomycin (Life Technologies). Transfections were carried out in 6-well plates using GeneJuice (Millipore) as per the manufacturer’s instructions. One million HEK 293T cells were stained in 100 μl of PBS at 4 °C for 15 min with primary antibody 3D12 for HLA-E (BioLegend) or 4D12 for Mamu-E (MBL), washed twice with PBS, stained with secondary antibody APC-conjugated goat-anti-mouse (H + L) F(ab')₂ fragment (Life Technologies), washed as before and fixed in 100 μl of Cytofix (BD Biosciences). Stained cells were acquired using a CyAn ADP Analyser (Beckman Coulter) and analyzed using FlowJo (TreeStar).

Approximately 150,000 cells per well were seeded on 6-well tissue culture plates, allowed to adhere and enter log phrase overnight. Each well was treated with the indicated agents for 2 days. Culture medium was collected. The cells were washed twice with PBSA and the PBSA was retained to collect non-adhered cells. The cells were trypsinised, washed and resuspended with their respective culture medium-PBSA mixture. Along with the non-adhered cells, cells were pelleted and washed with PBSA twice. The washed cell pellet was then resuspended in Annexin V-Alexa Fluor 647 (Life Technologies) and propidium iodide (Sigma-Aldrich) solution, and incubated in the dark at RT for 15 minutes. The stained cells were analysed using a CyAn ADP analyser (Beckman Coulter). AV-Alexa Fluor 647 was measured by excitation at 642 nm and emission detection at 665 nm. propidium iodide (PI) was measured by excitation at 405 nm and emission detection at 675 nm.

The BD FITC Annexin V Apoptosis Detection kit (BD PharMingen, San Diego, CA) was used to determine the effect of carprofen or mavacoxib treatment on the level of apoptosis. The Annexin V analysis allows monitoring of changes in necrosis, early apoptosis and late apoptosis. The assay was performed according to the manufacturer’s instructions. Briefly, cells were grown until 60-70% confluent and treated with either carprofen or mavacoxib at final concentrations of 50 μM and 100 μM. Untreated cells were used as controls for each cell line. Cells were treated for 24 h and 48 h at 37°C, 5% CO₂. Subsequently, cells were washed twice with cold PBS, trypsinized and resuspended in 1 ml of 0.1% BSA containing PBS. 1 × 10⁵ cells/ml and were then stained with Annexin V FITC and propidium iodide for 15 min in the dark. The CyAn ADP analyser (Beckman Coulter Inc. CA, USA) was used and the data was analyzed using Summit v4.3 software.

At 48 hrs post-doxycycline induction, 5 × 10⁵ cells per growth condition were pelleted at 200 × g, 5 min at room temperature and resuspended in at total volume of 83 μl (comprising 75 μl PhiPhiLux reagent and 8 μl FBS), and incubated for 30 min at 37 °C. Cells were washed in 1 ml PhiPhiLux buffer and pelleted as before. Cells were resuspended in buffer and fluorescence measured by flow cytometry on the FITC channel of a Cyan ADP analyser (Beckman Coulter). Data was analyzed using FlowJo software (TreeStar).

S2 cells were grown in six-well plates as described above and treated with vehicle alone or increasing concentrations of 9-(S)-HODE for 24 h. Cells were pelleted, resuspended in 1 ml 1× PBS, and fixed using 4% formaldehyde (Sigma-Aldrich) for 10 min. Cells were washed twice using 1× PBS, resuspended in 1 ml PI/RNase staining buffer (BD Pharmingen), and incubated at room temperature for 30 min before analysis on a Beckman Coulter Cyan ADP analyser. The S2 population was gated using forward scatter and side scatter and PI staining quantitated using 800 V setting. PI staining distribution was determined using Summit V4.3.02 analysis software.