5. Prepare lipoplexes. To prepare 500 μl of lipoplexes:
(a) For reagent A consisting of PTAI-6-8/PTAI-7 + DOPE.
– Dilute 3 μl of PTAI-6-8/PTAI-7 based reagent
prepared in Subheading 3.1 (step 4(a)) in 59.5 μl of
DMEM F-12 Ham.
– Add 62.5 μl of plasmid DNA solution at 0.08 μg/μl
concentration (diluted in DMEM F-12 Ham).
– Incubate 30 min at RT (20–25
C) with gentle
rocking.
– Add 375 μl of DMEM F-12 Ham (for DU145 cells) or
EMEM (for XC cells) and mix by gentle pipetting.
(b) For reagent B consisting of PTAI-10-14/PTAI11 + DOPE + DC-chol.
– Dilute 7.5 μl of PTAI-10-14/PTAI-11 based reagent
prepared in Subheading 3.1 (step 4(b)) in 55 μl of
DMEM F-12 Ham.
– Add 62.5 μl of plasmid DNA solution at 0.2 μg/μl
concentration (diluted in DMEM F-12 Ham).
– Incubate 30 min at RT (20–25
C) with gentle
rocking.
– Add 375 μl of DMEM F-12 Ham (for DU145 cells) or
EMEM (for XC cells) and mix by gentle pipetting.
6. Add lipoplexes to cells (see Note 8) in serum-free conditions or
in the presence of serum:
l
Serum-free lipofection: aspirate medium from cells
(optional: wash cells with medium without serum and antibiotics) and add the following:
(a) For reagent A consisting of PTAI-6-8/PTAI7 + DOPE.
– 200 μl of DMEM F-12 Ham (for DU145 cells) or
EMEM (for XC cells) and 200 μl of PTAI-6-8/
PTAI-7 based lipoplexes prepared in step 5(a).
(b) For reagent B consisting of PTAI-10-14/PTAI11 + DOPE + DC-chol.
– 293.3 μl of DMEM F-12 Ham (for DU145 cells) or
EMEM (for XC cells) and 106.7 μl of PTAI-10-14/
PTAI-11 based lipoplexes prepared in step 5(b).
l
Lipofection in the presence of serum: aspirate medium from
cells partially—leave 200 μl of medium in each well of
24-well plate (see Note 9) and add the following:
(c) For reagent A consisting of PTAI-6-8/PTAI7 + DOPE.
– 200 μl of PTAI-6-8/PTAI-7 based lipoplexes
prepared in step 5(a).
Lipofection-Based Delivery of DNA Vaccines
397
(a) For reagent A consisting of PTAI-6-8/PTAI-7 + DOPE.
– Dilute 3 μl of PTAI-6-8/PTAI-7 based reagent
prepared in Subheading 3.1 (step 4(a)) in 59.5 μl of
DMEM F-12 Ham.
– Add 62.5 μl of plasmid DNA solution at 0.08 μg/μl
concentration (diluted in DMEM F-12 Ham).
– Incubate 30 min at RT (20–25
C) with gentle
rocking.
– Add 375 μl of DMEM F-12 Ham (for DU145 cells) or
EMEM (for XC cells) and mix by gentle pipetting.
(b) For reagent B consisting of PTAI-10-14/PTAI11 + DOPE + DC-chol.
– Dilute 7.5 μl of PTAI-10-14/PTAI-11 based reagent
prepared in Subheading 3.1 (step 4(b)) in 55 μl of
DMEM F-12 Ham.
– Add 62.5 μl of plasmid DNA solution at 0.2 μg/μl
concentration (diluted in DMEM F-12 Ham).
– Incubate 30 min at RT (20–25
C) with gentle
rocking.
– Add 375 μl of DMEM F-12 Ham (for DU145 cells) or
EMEM (for XC cells) and mix by gentle pipetting.
6. Add lipoplexes to cells (see Note 8) in serum-free conditions or
in the presence of serum:
l
Serum-free lipofection: aspirate medium from cells
(optional: wash cells with medium without serum and antibiotics) and add the following:
(a) For reagent A consisting of PTAI-6-8/PTAI7 + DOPE.
– 200 μl of DMEM F-12 Ham (for DU145 cells) or
EMEM (for XC cells) and 200 μl of PTAI-6-8/
PTAI-7 based lipoplexes prepared in step 5(a).
(b) For reagent B consisting of PTAI-10-14/PTAI11 + DOPE + DC-chol.
– 293.3 μl of DMEM F-12 Ham (for DU145 cells) or
EMEM (for XC cells) and 106.7 μl of PTAI-10-14/
PTAI-11 based lipoplexes prepared in step 5(b).
l
Lipofection in the presence of serum: aspirate medium from
cells partially—leave 200 μl of medium in each well of
24-well plate (see Note 9) and add the following:
(c) For reagent A consisting of PTAI-6-8/PTAI7 + DOPE.
– 200 μl of PTAI-6-8/PTAI-7 based lipoplexes
prepared in step 5(a).
Lipofection-Based Delivery of DNA Vaccines
397