2.2 Invasive E. coli
Vector and Cell Lines
1. Bacterial growth media: Brain Heart Infusion (BHI) media.
Prepare according to manufacturer instructions (see Note 4)
and transfer to a 250 mL conical flask. Autoclave to sterilize.
Store at room temperature.
2. Invasive E. coli vector: E. coli DH10B (pGB2Ωinv-hly,
pEGFPN2) [2].
3. Fluorescent E. coli vector: E. coli DH10B (pGB2Ωinv-hly,
pUltraRFP-KM) [2, 10].
4. Noninvasive control vector: E. coli DH10B (pEGFPN2) [2].
5. Cell lines: SK-N-SH (ATCC #:), HT1080 (ATCC #:
CCL-121), GM02775 (Coriell Institute for Medical
Research), or other desired cells that express β1 integrin (see
Note 5).
2.3 Mammalian Cell
Invasion
2.3.1 Cell Culture Media
1. Invasion media: RPMI or DMEM containing 10 mM L-glutamine but not containing serum. Store at 4
C. Warm to 37
C
in a sterile water bath before us (see Note 6).
2. Complete culture media: RPMI or DMEM containing 10%
FBS and 10 mM L-glutamine. Store at 4
C. Warm to 37
C
in a water bath before use (see Note 7).
3. 0.05% Trypsin ETDA.
2.4 Cell Culture
Materials
1. Six-well cell culture plates.
2. T-75 Cell culture flask.
3. 5 mL serological pipettes.
4. 10 mL serological pipettes.
5. Automatic pipettor.
6. Plate centrifuge.
7. Trypan blue.
8. Hemocytometer.
9. 15 mL Falcon centrifuge tubes.
10. 50 mL Falcon centrifuge tubes.
11. Temperature controlled water bath.
12. Silica gel (see Note 8).
13. Thin tipped forceps.
2.5 Microscopy
1. Hitachi SU8010 scanning electron microscope.
2. Nikon eclipse TE2000-E/C1si.
3. Glass microscope slides.
4. Plastic microscope coverslips.
5. Glass microscope coverslips.
6. Lint-free tissue.
High-Resolution Imaging of Bacterial Invasion
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