4 Notes
1. Use molecular grade ethanol. Gradient solutions of EtOH can
be prepared in 50 mL centrifuge tubes to provide sufficient
volume to allow sample dehydration in multiple six-well plates.
Larger volumes can be prepared provided measurements are
kept precise. It is advisable that ethanol gradient solutions are
prepared fresh for each experiment.
2. Alternatively, 4% Paraformaldehyde (PFA) (stock) can be used.
Dissolve 5 g of PFA into 100 mL of 1Â PBS heated to 60
C
with stirring. Filter the solution when cooled. Dilute to 0.4%
working concentration in PBS. Store at À20
C. For rapid
preparation of invaded adherent cell populations, fixation
with 2.5% glutaraldehyde for 1 h at 37
C provides the most
stable results. However, rapid fixation may result in damage to
specimen including shrinkage of bacteria and or cracks on cell
membranes. Cells fixed with 0. 25% glutaraldehyde or 0.4%
PFA overnight at 4
C will result in better preserved cell
specimen with more intact membranes. Glutaraldehyde and
paraformaldehyde should be handled in a fume cabinet.
3. Application of acridine orange results in a fluorescent green
stain to membrane, cytoplasmic components, and intracellular
DNA. This should be considered when bacterial vectors constitutively expressing fluorescent proteins are used. Acridine
orange may be substituted with propidium iodide to stain the
same cellular components fluorescent red. Bacterial vectors not
expressing alternate fluorescent proteins such as GFP or RFP
will non-discriminatorily be stained by both fluorescent dyes.
4. Reconstituted BHI growth media should always be sterilized.
Media can be prepared and stored for future experiments but is
recommended to be made fresh.
5. Human Fabry disease fibroblasts (GM02775) are typically
cultured in MEM containing Earle’s salts and non-essential
amino acids supplemented with 15% FBS. They can tolerate
invasion media without serum for about 2 h before they begin
to shrink and detach from cell culture vessels. Cells that express
integrin are more permissive to attachment of invasive E. coli.
6. The base growth media used for each experiment is cell line
specific. Most immortalized cell lines are maintained in RPMI
or DMEM. It is important that media used for invasion does
not contain serum as this leads to clumping of the bacterial
vectors, which drastically reduces their attachment and invasion
capabilities.
7. Complete cell culture media used to promote recovery of cells
following the invasion process should contain all necessary
components including 2 mM L-glutamine and 10% FBS. It is
High-Resolution Imaging of Bacterial Invasion
37
1. Use molecular grade ethanol. Gradient solutions of EtOH can
be prepared in 50 mL centrifuge tubes to provide sufficient
volume to allow sample dehydration in multiple six-well plates.
Larger volumes can be prepared provided measurements are
kept precise. It is advisable that ethanol gradient solutions are
prepared fresh for each experiment.
2. Alternatively, 4% Paraformaldehyde (PFA) (stock) can be used.
Dissolve 5 g of PFA into 100 mL of 1Â PBS heated to 60
C
with stirring. Filter the solution when cooled. Dilute to 0.4%
working concentration in PBS. Store at À20
C. For rapid
preparation of invaded adherent cell populations, fixation
with 2.5% glutaraldehyde for 1 h at 37
C provides the most
stable results. However, rapid fixation may result in damage to
specimen including shrinkage of bacteria and or cracks on cell
membranes. Cells fixed with 0. 25% glutaraldehyde or 0.4%
PFA overnight at 4
C will result in better preserved cell
specimen with more intact membranes. Glutaraldehyde and
paraformaldehyde should be handled in a fume cabinet.
3. Application of acridine orange results in a fluorescent green
stain to membrane, cytoplasmic components, and intracellular
DNA. This should be considered when bacterial vectors constitutively expressing fluorescent proteins are used. Acridine
orange may be substituted with propidium iodide to stain the
same cellular components fluorescent red. Bacterial vectors not
expressing alternate fluorescent proteins such as GFP or RFP
will non-discriminatorily be stained by both fluorescent dyes.
4. Reconstituted BHI growth media should always be sterilized.
Media can be prepared and stored for future experiments but is
recommended to be made fresh.
5. Human Fabry disease fibroblasts (GM02775) are typically
cultured in MEM containing Earle’s salts and non-essential
amino acids supplemented with 15% FBS. They can tolerate
invasion media without serum for about 2 h before they begin
to shrink and detach from cell culture vessels. Cells that express
integrin are more permissive to attachment of invasive E. coli.
6. The base growth media used for each experiment is cell line
specific. Most immortalized cell lines are maintained in RPMI
or DMEM. It is important that media used for invasion does
not contain serum as this leads to clumping of the bacterial
vectors, which drastically reduces their attachment and invasion
capabilities.
7. Complete cell culture media used to promote recovery of cells
following the invasion process should contain all necessary
components including 2 mM L-glutamine and 10% FBS. It is
High-Resolution Imaging of Bacterial Invasion
37