helpful to increase the serum concentration to 12% following
the invasion process to improve recovery rates and limit cell
death.
8. We use silica gel to create transient desiccators to store and
transport processed coverslips containing dehydrated samples.
One teaspoon of silica gel can be placed in each well of a
six-well plate and coverslips can be rested on the gel beads.
Plates can be covered and air-sealed using parafilm to preserve
dehydrated samples for SEM.
9. When using plastic coverslips, slightly bending a corner of the
slips will enable much easier handling using forceps. Glass
coverslips are ideal for confocal microscopy, but do not easily
allow cells to attach. Higher cell numbers may be seeded to
provide the desired confluence of approximately 60–70% on
the day of invasion. We sterilize plastic and glass coverslips
using 70% EtOH, followed by UV irradiation for 30 min
prior to placement into sterile six-well plates.
10. Directly pipetting wash buffer onto coverslips may result in
unwanted cell detachment. This is more of a concern with glass
coverslips where cellular attachment to the coverslip is not as
robust as it is to plastic. Washing steps can be repeated to
ensure all traces of FBS are removed from the wells. Prolonged
exposure of cells to PBS will however affect their morphology
and may negatively impact the bacterial vectors ability to attach
during invasion.
11. Plates can be quickly viewed under a standard light microscope
to ensure that washing is thorough. Additional wash steps can
be performed to ensure that bacteria are completely removed
from areas of the culture surface that are not occupied by cells.
Alternatively, coverslips can be picked up with forceps and
gently dipped into 1Â PBS in a sufficiently deep container.
Multiple “wash basins” containing 1Â PBS can be set up for
the experiment to dip the coverslips in. It is however more
scalable to wash coverslips inside six-well plates using a serological pipette.
12. Samples can be fixed with stock concentrations of glutaraldehyde or paraformaldehyde for 1 h at 37
C, or alternatively
with working concentrations at 4
C overnight. Overnight
fixation provides better sample quality by leaving cell and bacteria membranes intact. The quicker samples are fixed, the
higher the likelihood of membrane shrinkage and cracking.
We have found that fixing cells immediately following the
invasion procedure allows the analysis of bacterial vector interaction with the cell membrane, prior to their internalization
into the cytoplasm. Cells invaded at 37
C for 1 h and fixed
immediately after will feature bacteria external to the cell.
38
Andrew N. Osahor et al.
the invasion process to improve recovery rates and limit cell
death.
8. We use silica gel to create transient desiccators to store and
transport processed coverslips containing dehydrated samples.
One teaspoon of silica gel can be placed in each well of a
six-well plate and coverslips can be rested on the gel beads.
Plates can be covered and air-sealed using parafilm to preserve
dehydrated samples for SEM.
9. When using plastic coverslips, slightly bending a corner of the
slips will enable much easier handling using forceps. Glass
coverslips are ideal for confocal microscopy, but do not easily
allow cells to attach. Higher cell numbers may be seeded to
provide the desired confluence of approximately 60–70% on
the day of invasion. We sterilize plastic and glass coverslips
using 70% EtOH, followed by UV irradiation for 30 min
prior to placement into sterile six-well plates.
10. Directly pipetting wash buffer onto coverslips may result in
unwanted cell detachment. This is more of a concern with glass
coverslips where cellular attachment to the coverslip is not as
robust as it is to plastic. Washing steps can be repeated to
ensure all traces of FBS are removed from the wells. Prolonged
exposure of cells to PBS will however affect their morphology
and may negatively impact the bacterial vectors ability to attach
during invasion.
11. Plates can be quickly viewed under a standard light microscope
to ensure that washing is thorough. Additional wash steps can
be performed to ensure that bacteria are completely removed
from areas of the culture surface that are not occupied by cells.
Alternatively, coverslips can be picked up with forceps and
gently dipped into 1Â PBS in a sufficiently deep container.
Multiple “wash basins” containing 1Â PBS can be set up for
the experiment to dip the coverslips in. It is however more
scalable to wash coverslips inside six-well plates using a serological pipette.
12. Samples can be fixed with stock concentrations of glutaraldehyde or paraformaldehyde for 1 h at 37
C, or alternatively
with working concentrations at 4
C overnight. Overnight
fixation provides better sample quality by leaving cell and bacteria membranes intact. The quicker samples are fixed, the
higher the likelihood of membrane shrinkage and cracking.
We have found that fixing cells immediately following the
invasion procedure allows the analysis of bacterial vector interaction with the cell membrane, prior to their internalization
into the cytoplasm. Cells invaded at 37
C for 1 h and fixed
immediately after will feature bacteria external to the cell.
38
Andrew N. Osahor et al.