serum free media. In addition, all traces of PBS should be
removed to avoid diluting the subsequently used invasion
media.
15. We find that extensive vortexing bacterial cultures after they
have been complexed with cationic reagents results in significant lysis of bacteria. This can result in false-positive gene
transfer resulting from free plasmids complexing with lipids
gaining access to cells.
16. In experiments that test multiple variables, 1.5 mL centrifuge
tubes containing mixtures of bacteria and reagents in invasion
media can become numerous and tedious to sort through. We
have found that the effects of these external reagents are not
altered if the reaction is scaled up into a 15 mL centrifuge tube
up to a maximum volume of 7 mL followed by aliquoting
accordingly into 6-well plates containing cells.
17. Wash steps should be executed carefully as some cell types are
prone to detachment. PBS should be gently pipetted on the
sides of the well and swirled carefully. Shrinkage/rounding of
cells occurs if PBS is not maintained at 37
C. Wash time
should be kept to a minimum and cells should not be exposed
to PBS for too long. We have found it is helpful in some cases
to condition cells to multiple washes with PBS during routine
maintenance/passaging to minimize detachment when washing during invasion.
18. Cells can be quickly viewed under a microscope after centrifugation to observe the attachment characteristics of bacteria to
the cell membrane.
19. Cells can be washed more than three times until all unattached
bacteria are removed. Invaded cell populations can be quickly
viewed under a microscope to determine is washing post-invasion is sufficient.
20. Gene expression can be observed approximately 24 h after
invasion. The maximum amount of gene expression however
is measurable between 48 and 72 h.
21. Cells should be thoroughly resuspended to prevent cell clumps
from blocking the fluidic systems of the flow cytometer.
22. Cells can be left on ice for up to 3 h away from direct light
before fluorescence signals begin to decrease. If cells are not
analyzed immediately, they should be resuspended again
immediately before they are analyzed using the flow cytometer.
23. A group of control cells that were not invaded with E. coli can
be used to establish a baseline for fluorescence measurements
in treated samples. We have found that fluorescence readings
are more accurate when control cells are subjected to the same
Enhancing Bactofection with Exogenous Reagents
25
removed to avoid diluting the subsequently used invasion
media.
15. We find that extensive vortexing bacterial cultures after they
have been complexed with cationic reagents results in significant lysis of bacteria. This can result in false-positive gene
transfer resulting from free plasmids complexing with lipids
gaining access to cells.
16. In experiments that test multiple variables, 1.5 mL centrifuge
tubes containing mixtures of bacteria and reagents in invasion
media can become numerous and tedious to sort through. We
have found that the effects of these external reagents are not
altered if the reaction is scaled up into a 15 mL centrifuge tube
up to a maximum volume of 7 mL followed by aliquoting
accordingly into 6-well plates containing cells.
17. Wash steps should be executed carefully as some cell types are
prone to detachment. PBS should be gently pipetted on the
sides of the well and swirled carefully. Shrinkage/rounding of
cells occurs if PBS is not maintained at 37
C. Wash time
should be kept to a minimum and cells should not be exposed
to PBS for too long. We have found it is helpful in some cases
to condition cells to multiple washes with PBS during routine
maintenance/passaging to minimize detachment when washing during invasion.
18. Cells can be quickly viewed under a microscope after centrifugation to observe the attachment characteristics of bacteria to
the cell membrane.
19. Cells can be washed more than three times until all unattached
bacteria are removed. Invaded cell populations can be quickly
viewed under a microscope to determine is washing post-invasion is sufficient.
20. Gene expression can be observed approximately 24 h after
invasion. The maximum amount of gene expression however
is measurable between 48 and 72 h.
21. Cells should be thoroughly resuspended to prevent cell clumps
from blocking the fluidic systems of the flow cytometer.
22. Cells can be left on ice for up to 3 h away from direct light
before fluorescence signals begin to decrease. If cells are not
analyzed immediately, they should be resuspended again
immediately before they are analyzed using the flow cytometer.
23. A group of control cells that were not invaded with E. coli can
be used to establish a baseline for fluorescence measurements
in treated samples. We have found that fluorescence readings
are more accurate when control cells are subjected to the same
Enhancing Bactofection with Exogenous Reagents
25