Isolation of Phagosomes Containing Magnetic Bead-Tagged Bacteria from Infected Macrophages

Zusammenfassung

Source: Gutiérrez, S., et al., Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages. J. Vis. Exp. (2017)

This video demonstrates the magnetic isolation of phagosomes containing biotin–streptavidin bead–coated bacteria from infected macrophages. The protocol includes buffer treatments, cell disruption, magnetic separation, and washing to yield purified phagosomes for analysis.

Protokoll

1. Isolation of S. typhimurium-containing Phagosomes
   1. Prepare the volume of required phagosome isolation buffer A (50 mM PIPES (Piperazine-N,N′-bis(2-ethanesulfonic acid)), pH.7, 50 mM MgCl₂, 5 mM EGTA (Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid) by adding dithiothreitol (DTT) to a final concentration of 1 mM, Cytochalasin B to a final concentration of 10 µM, and protease and phosphatase inhibitors as recommended by the manufacturer.
      NOTE: DTT and Cytochalasin B must be added to the phagosome isolation buffer A immediately before use. Cytochalasin B disrupts the cytoskeleton, facilitating the rupture of the cell membrane.
   2. At the desired time point, remove the medium from the infected cells and wash with sterile PBS (Phosphate-buffered saline) warmed to RT.
   3. Add 750 µL of phagosome isolation buffer A per dish and incubate 20 min on ice. When using different cell numbers, the volume of isolation buffer A should be adjusted proportionally.
   4. Add 250 µL of phagosome isolation buffer B (50 mM PIPES, pH.7, 50 mM MgCl₂, 5 mM EGTA, 220 mM Mannitol, and 68 mM sucrose). Rock the plate to ensure that the buffer reaches the complete surface of the dish. When using different cell numbers, the volume of isolation buffer B should be adjusted proportionally.
   5. Remove the cells from the dish by gently scraping using a rubber policeman and transfer them to a pre-chilled 1.5 mL tube.
   6. Pass the cell suspension through a 26G needle using a 1 mL syringe at least 15 times (one aspiration plus one ejection of the cell suspension counts as one time). This is enough to release the cytosolic content of BMDMs (Bone marrow-derived macrophages). The number of passes through the needle must be optimized for every cell type.
   7. Place the cell suspension on the magnetic rack and wait 5 min. The particles attached to the magnet are the phagosomes containing coated-S. typhimurium. The suspension contains the rest of the cellular components.
   8. Transfer the suspension into a 1.5 mL tube labeled as "cytosol".
   9. Remove the tube with the isolated phagosomes from the magnetic rack and resuspend them in 1 mL of sterile PBS.
   10. Place the phagosome suspension on the magnetic rack, wait for 5 min, and remove the PBS.
   11. Repeat wash of the isolated S. typhimurium-containing phagosomes.
   12. Finally, remove the PBS and resuspend the phagosomes in the required buffer, such as a radioimmunoprecipitation assay (RIPA) buffer, for protein analysis. Approximately 50-200 µg of protein is obtained per sample following this protocol, depending on the initial amount of cells and the MOI (Multiplicity of infection) of infection.

Materialien

Liste der in diesem Artikel verwendeten Materialien

Name	Unternehmen	Katalognummer	Kommentare
EZ-Link NHS Biotin	Thermo Fisher Scientific	20217
FluidMag Streptavidin	Chemicell	4205
PIPES (Piperazine-N,N′-bis(2-ethanesulfonic acid)	Carl Roth	9156.2
MgCl₂	Carl Roth	A537.4
EGTA (Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid)	Carl Roth	3054.3
Sucrose	Carl Roth	4621.1
Mannitol	Carl Roth	4175.1
DTT (Dithiothreitol )	Sigma	43816
Halt Protease and Phosphatase inhibitor cocktail	Thermo Fisher Scientific	1861280
Cytochalasin B	Sigma	C6762
DYNAL or DynaMag Magnet	Thermo Fisher Scientific	12321D
Salmonella enterica serovar typhimurium SL1344	Leibniz Institute DSMZ-German collection of Microorganisms and Cell Cultures
RPMI (Roswell Park Memorial Institute)	Biochrom	FG1415
PBS (Phosphate-buffered saline)	Biochrom	L1825
Cy5-streptavidin	Invitrogen	SA1011