Figure 1

Overview of method. 1. "Magnetic bacteria" are prepared by covalently attaching very small magnetite particles to the surface of the bacteria. This can be done in large batches. If dead bacteria are used the finished product may be stored for several weeks at 4°C. 2. Synchronized phagocytosis of the magnetic bacteria is achieved through a 30-s centrifugation of a mixture of phagocytic cells and magnetic bacteria. This step may be repeated after resuspension to increase the interaction efficiency. Simultaneous phagocytosis of multiple samples can be performed using multi-channel pipettes in conjunction with either test tubes or microtiter plates. 3. After completed presentation, free bacteria are washed away. Following an optional chase period, the suspension is then put on ice and pooled, and the buffer changed to an isotonic sucrose solution containing protease inhibitors and DNAse. The resulting suspension is put in a bomb cylinder and subjected to nitrogen cavitation (300 psi, 10 min) to disrupt the phagocytic cells. 4. Aliquots of cell lysate are put into microtiter wells. Phagosomes are retrieved magnetically using a magnetic rod. Each well is probed several times to increase yield. 5. Phagosome integrity is determined using direct fluorescent staining of a phagosome membrane marker and antibodies recognizing free or partially free bacteria. 6. Isolated phagosomes are analyzed using immunofluorescence microscopy, flow cytometry, or immunoblot. Steps 2–5 can be achieved in less than 1 h.