Cytomics fc 500 analyzer

Cultured bacteria and bacteroids were fixed in 90% ethanol overnight at −20°C. Cells were then washed twice with PBS followed by centrifugation for 2 min at 1,200 × g. Pelleted cells were stained with propidium iodide (PI)-RNase staining buffer solution (BD Biosciences, 550825) for 30 min at room temperature. For each flow cytometry experiment, the DNA content was measured in a population of 20,000 cells with a Cytomics FC500 analyzer (Beckman Coulter Ltd.). Data analysis was performed with CXP software (Beckman Coulter Ltd.) (Wake and Errington, 1995 (link)).

The tvb^s1/s1, tvb^s1/r3 and tvb^r3/r3 CEFs were seeded in triplicate wells at a density of 2×10⁵ per well in a 6-well plate. After reaching about 90% confluence, CEFs of defined origin were harvested by 0.25% trypsin solution, and washed in PBS supplemented with 2% calf serum (PBS-CS), centrifuged for 5 min at 500×g, and resuspended in 200 μL of PBS-CS. The cells in PBS-CS were incubated with supernatant containing SU(B)-rIgG, SU(D)-rIgG or SU(E)-rIgG fusion protein with the same concentration (10 ng of rIgG Fc fragment per ml) on ice for 1 h. After three washes with PBS-CS, the goat anti-rabbit IgG linked to Alexa Fluor 488 (Invitrogen) was diluted 1:100 in PBS supplemented with 4% calf serum, and washed cells were incubated in 500 μL of diluted antibody on ice for 30 min. After incubation and three washes in PBS-CS, the complexes of cells with immunoadhesins were resuspended in 200 μL PBS-CS, and the percentage of fluorescence-positive cells was quantitated by FACS using a Cytomics FC 500 analyzer (Beckman Coulter, Kurt, U.S.A).