with a defined length of polyprenyl chain of 7 or 11 isoprene
units, respectively. Natural mixtures of polyprenols are easier to
obtain (they are isolated from plant material) than single polyprenols (have to be separated from the mixtures) and thus
cheaper, while having the same lipofecting efficacy.
2. Bacterial cultures should be grown under antibiotic selection to
prevent loss of plasmid.
3. In order to remove endotoxin from glassware, it should be
sterilized at 180
C overnight.
4. PTAI and DOPE should easily dissolve in ethanol at the indicated concentrations at room temperature (RT—20–25
C). If
DOPE is not dissolved completely (e.g., after storage in the
freezer), place it in a shaker at 37
C for 10 min. To dissolve
DC-cholesterol, warm up the solution in a water bath to
around 50
C and vortex. Store dissolved lipids in glass bottles
with Teflon seals at À20
C.
5. Mixture of lipids in ethanol should be injected into medium
very rapidly and mixed by intense pipetting.
6. Every time before use, reagents should be taken out of the 4
C
storage conditions in advance and brought to RT (20–25
C).
Immediately before use, reagents should be vortexed at the
highest vortex settings for 3 min at RT (20–25
C). When
stored at 4
C, reagents are stable for at least a week. Do not
freeze the reagents.
7. To ensure proper confluency and even cell distribution, prepare
a homogenous stock of cells in the medium and plate them to
every well from this stock solution. After seeding cells, gently
slide the plate in a cross-like pattern (forward and backward and
side-to-side) or in a figure-eight-like pattern three to four
times. If you are planning to perform your experiment in a
plate with very small wells (e.g., 48- or 96-wells), you may not
transfer the plate to the incubator immediately but leave them
under the hood for up to an hour after seeding to let cells
attach to the surface. A proper amount of medium (not too
small, recommended by the manufacturer) for the dish/well
should also be used; for example, for a 24-well plate, it should
be not less than around 400 μl/well. This prevents meniscus
effect/rings (increased cell density along the sides of the
dishes), as the curve of the meniscus along the sides of the
vessel is deeper and contains more medium and cells per unit
surface area than the media-poor area in the center of the dish/
well.
8. It is very important to add medium to cells first and then add
lipoplexes. Add lipoplexes dropwise to the whole area of the
well. Do not touch the sides of the well when adding
lipoplexes.
Lipofection-Based Delivery of DNA Vaccines
401
units, respectively. Natural mixtures of polyprenols are easier to
obtain (they are isolated from plant material) than single polyprenols (have to be separated from the mixtures) and thus
cheaper, while having the same lipofecting efficacy.
2. Bacterial cultures should be grown under antibiotic selection to
prevent loss of plasmid.
3. In order to remove endotoxin from glassware, it should be
sterilized at 180
C overnight.
4. PTAI and DOPE should easily dissolve in ethanol at the indicated concentrations at room temperature (RT—20–25
C). If
DOPE is not dissolved completely (e.g., after storage in the
freezer), place it in a shaker at 37
C for 10 min. To dissolve
DC-cholesterol, warm up the solution in a water bath to
around 50
C and vortex. Store dissolved lipids in glass bottles
with Teflon seals at À20
C.
5. Mixture of lipids in ethanol should be injected into medium
very rapidly and mixed by intense pipetting.
6. Every time before use, reagents should be taken out of the 4
C
storage conditions in advance and brought to RT (20–25
C).
Immediately before use, reagents should be vortexed at the
highest vortex settings for 3 min at RT (20–25
C). When
stored at 4
C, reagents are stable for at least a week. Do not
freeze the reagents.
7. To ensure proper confluency and even cell distribution, prepare
a homogenous stock of cells in the medium and plate them to
every well from this stock solution. After seeding cells, gently
slide the plate in a cross-like pattern (forward and backward and
side-to-side) or in a figure-eight-like pattern three to four
times. If you are planning to perform your experiment in a
plate with very small wells (e.g., 48- or 96-wells), you may not
transfer the plate to the incubator immediately but leave them
under the hood for up to an hour after seeding to let cells
attach to the surface. A proper amount of medium (not too
small, recommended by the manufacturer) for the dish/well
should also be used; for example, for a 24-well plate, it should
be not less than around 400 μl/well. This prevents meniscus
effect/rings (increased cell density along the sides of the
dishes), as the curve of the meniscus along the sides of the
vessel is deeper and contains more medium and cells per unit
surface area than the media-poor area in the center of the dish/
well.
8. It is very important to add medium to cells first and then add
lipoplexes. Add lipoplexes dropwise to the whole area of the
well. Do not touch the sides of the well when adding
lipoplexes.
Lipofection-Based Delivery of DNA Vaccines
401