Accuri cytometer

A flow cytometry approach was applied to determine cell apoptosis and cell cycle. An annexin V-FITC/propidium iodide cell apoptosis detection kit (BD Biosciences, USA) was used to detect cell apoptosis. U251 and U87 cells were trypsinized and re-suspended in 500 μL binding buffer, then 1 μL annexin V-FITC and 1 μL propidium iodide were added to the cells for 25 minutes of incubation. Cells were washed once with the binding buffer and then analyzed on the Accuri Cytometer (BD Biosciences). The cell cycle assay kit (Beyotime, China) was used to assess the cell cycle. U251 and U87 cells were fixed with 75% anhydrous ethanol overnight in the fridge. After RNase digestion, the samples were stained using propidium iodide for 30 minutes in the dark and then analyzed on the Accuri Cytometer (BD Biosciences).

Neutrophils were separated from whole blood of runners on Histopaque-1077 (d = 1.077) gradients (Sigma-Aldrich, St. Louis, MO, USA) (Boyum et al. 1968). The neutrophils obtained from the blood sample athletes were counted in a Neubauer chamber under an optical microscope (Nikon, Melville, NY, USA). Neutrophils (1x10⁶ cells / ml) were incubated with antibody conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE) or allophycocyanin (APC) for 30 minutes in the dark at room temperature. The expression of surface molecules ICAM-1 (CD54—FITC), L-selectin (CD62—FITC), TNFR-1 receptor (CD120b—PE) and Fas (CD95 –APC) were analyzed by flow cytometry. The fluorescence was determined by flow cytometry at wavelengths 530/30 nm (FITC) 660/20 nm (APC) or 695/40 nm (PE) (BD Accuri cytometer) and ten thousand events per sample were acquired in histograms.
DNA fragmentation was analyzed by flow cytometry (BD Accuri cytometer) after DNA staining with propidium iodide, according to the method described by Nicoletti et al. (1991) [18 (link)].

To determine if VLP-based therapies can induce immune cell proliferation, PBMCs from different non-allergic volunteers were isolated using a Lymphoprep density gradient. PBMCs were then seeded in flat-bottom 96-well plates at a concentration of 2·10⁵ cells/mL and incubated with VLP-Pru p 3 or VLP-Complex (20 ng/µL VLP; 5 ng/µL Pru p 3 or complex). Complex (5 ng/µL) was used as a positive control to induce proliferation. After 5 days (37°C, 5% CO₂), cellular concentration was calculated using a BD Accuri cytometer. SI for each stimulus was calculated as the variation of cell concentration over time normalized with the variation of the control.
To evaluate the immunotoxicity of the formulations, human monocytes (THP1 cells; In vivogen) were seeded in flat-bottom 96-well plates at a concentration of 1·10⁶ cells/mL and incubated with the same stimuli as described above. 10-hidroxycamptothecin (0.25 ng/µL) was used as a positive control to induce apoptosis. After 24 h, cells were stained with annexin V-FITC (1:100, Merck) and PI (1:100, Merck) for 10 min (RT). Samples were analyzed using a BD Accuri cytometer and results were processed using FCS Express 7 Plus software. Unstained samples were used as background controls.