the invasion process [9], providing clear details of successful vector
attachment events on the target cell membrane, and well-defined
indications of the internalization of the vector into the cytoplasm.
Culturing cells on microscope coverslips inserted into typical
culture vessels allows invaded cell populations to be prepared for
advanced microscopy with minimal effort. Using SEM, the interactions of invasive vectors with the cell membrane can be studied
with high enough resolution to allow the quantification of individual successful bacterial attachment events to the cell. This procedure can be performed without the use of hazardous and expensive
fixatives such as osmium tetroxide or hexamethyldisilane, as well as
critical point or freeze drying [9].
Invaded populations on culture slips can also be subjected to
confocal microscopy to clearly determine vector internalization
into the cytoplasm. This process can be achieved using inexpensive
fluorescent stains meant for DNA staining such as acridine orange
[11] and propidium iodide [12] to demarcate the cell membrane
and nucleus, allowing distinction between vectors constitutively
expressing fluorescent proteins that are external to the cell membrane, and vectors that have been internalized into the cytoplasm.
These techniques take advantage of easy preparatory steps and
affordable materials to appropriately visualize important aspects of
the invasion process when bacteria are used as vectors for molecule
delivery. These methods may be applicable to precisely evaluate the
effectiveness of the membrane adherence and internalization characteristics of other bacterial vectors.
2 Materials
Prepare all solutions using ultrapure water and analytical grade
reagents. Prepare and store all reagents at room temperature
(unless indicated otherwise). Sterilize all reagents and culture vessels to avoid contamination. Diligently follow all waste disposal
regulations when disposing waste materials.
2.1 Buffers and
Reagents
1. Wash buffer: Phosphate Buffered Saline (PBS). Dilute stock
from 10Â to 1Â using sterilized dH 2 O. Store at 4
C. Warm to
37
C before use during bacterial invasion procedures.
2. Dehydration Buffers: Dilute ethanol (EtOH) in distilled water
to concentrations of 25, 40, 60, 80, 90, and 100% (see Note 1).
3. Fixing solution: 2.5% glutaraldehyde (stock): Dilute to 0.25%
working concentration in PBS (see Note 2).
4. 10 mg/mL Acridine orange fluorescent stains: Dilute in 1Â
PBS to a working concentration of 20 μg/mL. Alternatively,
50 μg/mL Propidium Iodide (stock). Dilute in 1Â PBS to a
working concentration of 1 μg/mL (see Note 3).
32
Andrew N. Osahor et al.
attachment events on the target cell membrane, and well-defined
indications of the internalization of the vector into the cytoplasm.
Culturing cells on microscope coverslips inserted into typical
culture vessels allows invaded cell populations to be prepared for
advanced microscopy with minimal effort. Using SEM, the interactions of invasive vectors with the cell membrane can be studied
with high enough resolution to allow the quantification of individual successful bacterial attachment events to the cell. This procedure can be performed without the use of hazardous and expensive
fixatives such as osmium tetroxide or hexamethyldisilane, as well as
critical point or freeze drying [9].
Invaded populations on culture slips can also be subjected to
confocal microscopy to clearly determine vector internalization
into the cytoplasm. This process can be achieved using inexpensive
fluorescent stains meant for DNA staining such as acridine orange
[11] and propidium iodide [12] to demarcate the cell membrane
and nucleus, allowing distinction between vectors constitutively
expressing fluorescent proteins that are external to the cell membrane, and vectors that have been internalized into the cytoplasm.
These techniques take advantage of easy preparatory steps and
affordable materials to appropriately visualize important aspects of
the invasion process when bacteria are used as vectors for molecule
delivery. These methods may be applicable to precisely evaluate the
effectiveness of the membrane adherence and internalization characteristics of other bacterial vectors.
2 Materials
Prepare all solutions using ultrapure water and analytical grade
reagents. Prepare and store all reagents at room temperature
(unless indicated otherwise). Sterilize all reagents and culture vessels to avoid contamination. Diligently follow all waste disposal
regulations when disposing waste materials.
2.1 Buffers and
Reagents
1. Wash buffer: Phosphate Buffered Saline (PBS). Dilute stock
from 10Â to 1Â using sterilized dH 2 O. Store at 4
C. Warm to
37
C before use during bacterial invasion procedures.
2. Dehydration Buffers: Dilute ethanol (EtOH) in distilled water
to concentrations of 25, 40, 60, 80, 90, and 100% (see Note 1).
3. Fixing solution: 2.5% glutaraldehyde (stock): Dilute to 0.25%
working concentration in PBS (see Note 2).
4. 10 mg/mL Acridine orange fluorescent stains: Dilute in 1Â
PBS to a working concentration of 20 μg/mL. Alternatively,
50 μg/mL Propidium Iodide (stock). Dilute in 1Â PBS to a
working concentration of 1 μg/mL (see Note 3).
32
Andrew N. Osahor et al.