4. 10 mM Chloroquine: Dissolve 51.6 mg in 10 mL dH 2 O
(stock), working concentration of 50 μM (store at À20
C).
5. 12.5 mg/mL Tetracycline hydrochloride: Dissolve 12.5 mg in
10 mL in EtOH (stock), working concentration of 1 μg/mL
(store at À20
C). Keep away from light.
6. 10 mg/mL Polymyxin B sulfate: Dissolve 10 mg in dH 2 O
(stock), working concentration of 1 μg/mL (store at À20
C).
2.3 Mammalian Cell
Invasion
2.3.1 Cell Culture Media
1. Invasion media: RPMI or DMEM containing 10 mM L-glutamine. Store at 4
C. Warm to 37
C in a sterile water bath
before use (see Note 4).
2. Complete culture media: RPMI or DMEM containing 10%
Fetal Bovine Serum (FBS) and 10 mM L-glutamine. Store at
4
C. Warm to 37
C in a water bath before use (see Note 5).
2.3.2 Buffers and
Reagents
1. Wash buffer: Phosphate Buffered Saline. Dilute stock from
10Â to 1Â using sterilized dH 2 O. Store at 4
C. Warm to
37
C in a water bath before use.
2. 0.05% Trypsin ETDA.
3. 10 mg/mL Gentamicin. Store at 4
C.
Fig. 3 Polar and diffuse attachment of E. coli–lipid complexes to HT1080 cell membranes. Electron
microscope micrographs showing HT1080 fibroblasts (false colored pink) and invasive E. coli (false colored
blue) in lipid directed arrangements onto individual cells and distributed in the same manner in multiple cells
when Lipofectamine and PULSin are complexed with invasive E. coli to invade cells after 1 h of incubation,
compared to the attachment of invasive E. coli alone without lipids. Images are qualitative representations of a
three biological replicates for each treatment
Enhancing Bactofection with Exogenous Reagents
19
(stock), working concentration of 50 μM (store at À20
C).
5. 12.5 mg/mL Tetracycline hydrochloride: Dissolve 12.5 mg in
10 mL in EtOH (stock), working concentration of 1 μg/mL
(store at À20
C). Keep away from light.
6. 10 mg/mL Polymyxin B sulfate: Dissolve 10 mg in dH 2 O
(stock), working concentration of 1 μg/mL (store at À20
C).
2.3 Mammalian Cell
Invasion
2.3.1 Cell Culture Media
1. Invasion media: RPMI or DMEM containing 10 mM L-glutamine. Store at 4
C. Warm to 37
C in a sterile water bath
before use (see Note 4).
2. Complete culture media: RPMI or DMEM containing 10%
Fetal Bovine Serum (FBS) and 10 mM L-glutamine. Store at
4
C. Warm to 37
C in a water bath before use (see Note 5).
2.3.2 Buffers and
Reagents
1. Wash buffer: Phosphate Buffered Saline. Dilute stock from
10Â to 1Â using sterilized dH 2 O. Store at 4
C. Warm to
37
C in a water bath before use.
2. 0.05% Trypsin ETDA.
3. 10 mg/mL Gentamicin. Store at 4
C.
Fig. 3 Polar and diffuse attachment of E. coli–lipid complexes to HT1080 cell membranes. Electron
microscope micrographs showing HT1080 fibroblasts (false colored pink) and invasive E. coli (false colored
blue) in lipid directed arrangements onto individual cells and distributed in the same manner in multiple cells
when Lipofectamine and PULSin are complexed with invasive E. coli to invade cells after 1 h of incubation,
compared to the attachment of invasive E. coli alone without lipids. Images are qualitative representations of a
three biological replicates for each treatment
Enhancing Bactofection with Exogenous Reagents
19