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

Abstract

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.

Protocol

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 b....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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