Fsx100 system

Cells were fixed in 4% paraformaldehyde-PBS solution. For visualization of intracellular markers, cells were permeabilized with 0.1% Triton X-100-PBS solution, blocked with 2% BSA-PBS solution for 1 hr at room temperature, and incubated overnight at 4°C with primary antibodies as listed in Table S4. Appropriate fluorescence-tagged secondary antibodies (Molecular Probes and Vector Laboratories) were used for visualization. Cells were mounted in DAPI mounting medium (Vector Laboratories), and images were obtained using an Olympus IX71 microscope equipped with a DP71 digital camera or Olympus FSX100 system. MetaMorph Microscopy Automation and Image Analysis Software (Molecular Devices) were used to count positive cells in seven to ten randomly selected images at a final magnification of ×200 from each of three independent experiments. For flow cytometry, cells were dissociated into single cells using GIBCO Cell Dissociation Buffer (Invitrogen) and incubated in 1% BSA-PBS solution with the appropriate antibodies as listed in Table S4. For unconjugated primary antibodies, the cells were incubated with Alexa-Flour 488-conjugated anti-mouse IgG/IgM (Molecular Probes) for raising suitable secondary antibodies. Flow cytometry was performed using FACSCalibur (BD Biosciences) and analyzed using WinMDI 2.8 or FACSVerse (BD Biosciences) and FlowJo software.