8. Gently add 5 mL of complete media to the culture vessel using
a serological pipette to deactivate trypsin.
9. Collect detached cells into a 15 mL Falcon tube using a serological pipette. Centrifuge the cells at 225 Â g for 5 min at
room temperature.
10. Gently aspirate the supernatant using a serological pipette.
11. Resuspend the cell pellet in 1 mL of complete cell culture
media. Pipette gently to ensure homogenous resuspension.
Do not vortex.
12. Remove 10 μL of cells onto a hemocytometer using a micropipette and mix with 10 μL trypan blue dye to obtain a viable cell
count.
13. Prepare cells for invasion by seeding HT1080, SK-N-SH, or
GM02775 cells in 6-well plates containing plastic or glass
coverslips at a density of 1 Â 10
5 cells per well 24 h before
invasion experiments (see Note 9).
14. On the day of invasion, remove growth media from the cell
culture vessel using a sterile serological pipette and rinse cells
twice with 1Â PBS that has been prewarmed to 37
C using a
sterile water bath.
15. Remove 1Â PBS from culture vessel using a serological pipette
immediately before invasion.
3.3 Invasion
Procedure
1. Place sterile plastic or glass coverslips at the bottom of a six-well
plate. Seed cells into wells 24 h prior to invasion experiments by
dispensing cells resuspended in complete culture media into
wells using a serological pipette. Incubate at 37
C with 5%
CO 2 overnight (see Note 9).
2. On the day of invasion, discard spent culture media by aspiration using a serological pipette. Wash wells with 3 mL of 1Â
PBS by gently pipetting onto the side of wells (see Note 10).
3. Transfer bacterial cultures resuspended in invasion media onto
cells grown on microscope coverslips in six-well plates. Centrifuge the plates at 225 Â g for 10 min. Transfer the cells to a
37
C in a CO 2 incubator for 1 h (Narayanan et al. 2013).
4. Remove bacteria that are not attached to cell membranes by
washing wells three times with 3 mL of 1Â PBS using a serological pipette (see Note 11).
3.4 Sample
Preparation for
Scanning Electron
Microscopy
1. Fix cells by adding 2 mL of fixation buffer to each well using a
serological pipette and incubate plates at 4
C overnight (see
Note 12).
2. Aspirate fixing solution using a serological pipette. Wash the
wells and coverslips three times with 1Â PBS that has been
High-Resolution Imaging of Bacterial Invasion
35
a serological pipette to deactivate trypsin.
9. Collect detached cells into a 15 mL Falcon tube using a serological pipette. Centrifuge the cells at 225 Â g for 5 min at
room temperature.
10. Gently aspirate the supernatant using a serological pipette.
11. Resuspend the cell pellet in 1 mL of complete cell culture
media. Pipette gently to ensure homogenous resuspension.
Do not vortex.
12. Remove 10 μL of cells onto a hemocytometer using a micropipette and mix with 10 μL trypan blue dye to obtain a viable cell
count.
13. Prepare cells for invasion by seeding HT1080, SK-N-SH, or
GM02775 cells in 6-well plates containing plastic or glass
coverslips at a density of 1 Â 10
5 cells per well 24 h before
invasion experiments (see Note 9).
14. On the day of invasion, remove growth media from the cell
culture vessel using a sterile serological pipette and rinse cells
twice with 1Â PBS that has been prewarmed to 37
C using a
sterile water bath.
15. Remove 1Â PBS from culture vessel using a serological pipette
immediately before invasion.
3.3 Invasion
Procedure
1. Place sterile plastic or glass coverslips at the bottom of a six-well
plate. Seed cells into wells 24 h prior to invasion experiments by
dispensing cells resuspended in complete culture media into
wells using a serological pipette. Incubate at 37
C with 5%
CO 2 overnight (see Note 9).
2. On the day of invasion, discard spent culture media by aspiration using a serological pipette. Wash wells with 3 mL of 1Â
PBS by gently pipetting onto the side of wells (see Note 10).
3. Transfer bacterial cultures resuspended in invasion media onto
cells grown on microscope coverslips in six-well plates. Centrifuge the plates at 225 Â g for 10 min. Transfer the cells to a
37
C in a CO 2 incubator for 1 h (Narayanan et al. 2013).
4. Remove bacteria that are not attached to cell membranes by
washing wells three times with 3 mL of 1Â PBS using a serological pipette (see Note 11).
3.4 Sample
Preparation for
Scanning Electron
Microscopy
1. Fix cells by adding 2 mL of fixation buffer to each well using a
serological pipette and incubate plates at 4
C overnight (see
Note 12).
2. Aspirate fixing solution using a serological pipette. Wash the
wells and coverslips three times with 1Â PBS that has been
High-Resolution Imaging of Bacterial Invasion
35