Isolation of Intracellular Pathogenic Bacteria from Infected Host Cells

Abstract

Source: Sigal, N., et al. RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells. J. Vis. Exp. (2016).

This video demonstrates the isolation of internalized pathogenic bacteria from infected bone marrow-derived macrophages (BMDMs). Bacteria are allowed to attach to host cells, and unattached bacteria are washed away. After internalization, an antibiotic is added to eliminate non-internalized bacteria. The host cells are lysed, centrifuged, and the supernatant is filtered to capture the bacteria on filter paper, which is flash-frozen and stored for further use.

Protocol

NOTE: During the entire experiment, macrophage cells are incubated at 37 °C in a 5% CO₂ forced-air incubator and taken out of the incubator only for experimental manipulations, which are performed in a Class II biological safety cabinet. Working with Listeria monocytogenes bacteria is according to biological safety level 2 regulations.

1. Cell Preparation and Bacterial Infection (Days 1 and 2)

1. Day 1
   1. Seed 2.0 x 10⁷ bone marrow-derived macrophage cells (BMDM) on a 145 mm dish in 30 ml BMDM + Pen-Strep media (Table 2). Seed 3 plates for each bacterial ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Listeria monocytogenes 10403S
Bone marrow derived macrophages prepared from C57B/6 female mice
H₂O, RNAse free	Thermo Scientific	10977-015	DEPC-treated water can be used
DMEM	Gibco	41965039
Glutamine	Gibco	25030081
Sodium pyruvate	Gibco	11360-088
β-Mercaptoethanol	Gibco	31350010
Pen/Strep	Gibco	15140-122
Gentamicin	Sigma-Aldrich	G1397
FBS	Gibco	10270106
Dulbecco’s Phosphate Buffered Saline-PBS	Sigma-Aldrich	D8537
Brain heart infusion (BHI)	Merckmillipore	1104930500
Sodium acetate	Sigma-Aldrich	W302406
EDTA	Sigma-Aldrich	EDS
Ethanol absolute	Merck Millipore	1070174000
37 °C, 5% CO₂ forced-air incubator	Thermo Scientific	Model 3111
Cell scrapers	Nunc	179693
Kontes glass holder for 45 mm filters	Fisher	K953755-0045
MF-Millipore filters 45 mm, 0.45 µm	Merck Millipore	HAWP04700
SpeedVac system	Thermo Scientific	SPD131DDA
Vortex-Genie 2	Scientific Industries	Model G560E
145 mm cell culture dishes	Greiner	639 160
1.7 ml tubes, RNase-free	Axygen	MCT-175-C
4 °C table centrifuge	Eppendorf	5417R
Sterile pipettes, 25 ml	Greiner
Falcon tubes, 50 ml	Greiner