Fitc conjugated phalloidin

The expression and cellular location of both Bcl-2 and Bax in the HT-29 and Caco-cell lines were determined by immunofluorescence microscopy. Cells were grown on coverslips (50 000 cells per coverslip), allowed to adhere overnight and exposed to the test nucleosides and camptothecin for 6 hours. After rinsing with PBS, cells were fixed with 3% formaldehyde in PBS for 20 minutes. The cells were rinsed and permeabilized for 5 minutes with 0.25% Triton X100, prepared in PBS with 0.5% bovine serum albumin (BSA). Following permeabilization, cells were blocked with 1% (BSA) in PBS for 1 hour. Thereafter cells were washed and incubated overnight with the respective primary antibodies (Bcl-2 or Bax in PBS with 0.5% BSA) at 4°C. Mouse anti-Bcl-2 and mouse anti-Bax was obtained from Biovision and used at a concentration of 10 mg/mL. After washing with PBS, cells were incubated with species compatible Alexa-fluor 568 (Abcam) secondary antibody. FITC-conjugated phalloidin, (Abcam), was used to visualize the cytoskeleton and the nuclei were visualized with Hoechst 33342 stain as described previously. The cells were viewed using an Olympus BX41 epifluorescence microscope with the appropriate filters for each fluorochrome. Images were captured with an Olympus DP72 camera and analysed with the Olympus CellSens Software package.

Periosteal cells prepared as described above were fixed with 10% formalin and treated with phycoerythrin (PE)-conjugated Annexin V (BioLegend, San Diego, CA, USA) and fluorescein isothiocyanate (FITC)-conjugated phalloidin (Abcam, Cambridge, MA, USA) according to the manufacturers’ instructions. It is noted that Annexin V can detect phosphatidylserine in live or fixed cells [17 ]. The cells were examined am Eclipse 80i fluorescence microscope (Nikon).

Isolated PMNs (500.000 cells per measurement) were stimulated with fMLP (100 ng/ml) or left untreated for 5, 15, 30, 60, 120 and 180 sec. For every time point, regardless of stimulation, reaction was stopped by adding a Fixation Buffer (BioLegend) for 20 min at room temperature. Thereafter, cells were permeabilized and blocked to avoid non-specific binding. Following 20 min incubation, cells were intracellularly stained with FITC-conjugated Phalloidin (Abcam, 1:1000). As negative control, PMNs were left either unstimulated or unstained. Following 30 min incubation in the dark at 4°C, actin polymerization was determined by the level of Phalloidin expression, as measured by flow cytometry.