(d) For reagent B consisting of PTAI-10-14/PTAI11 + DOPE + DC-chol.
– 93.3 μl of DMEM F-12 Ham (for DU145 cells) or
EMEM (for XC cells) and 106.7 μl of PTAI-1014/PTAI-11 based lipoplexes prepared in step 5
(b).
7. After adding lipoplexes, gently slide the plate in a cross-like
pattern (forward and backward and side-to-side) or in a figureeight-like pattern three to four times.
8. Incubate cells at 37
C with 5% CO 2 for 5 h.
9. Add 400 μl of DMEM F-12 Ham (for DU145 cells) or EMEM
(for XC cells) supplemented with 20% FCS or FBS and
200 IU/ml penicillin, 20 μg/ml neomycin, and 20 μg/ml
streptomycin.
10. Verify transfection efficiency 24 h after transfection (see Note
10) by fluorescence microscopy or flow cytometry. Examples
of PTAI efficiency as components of lipofecting mixtures used
in vitro are shown in Fig. 2b and c.
Optional: for fluorescent microscopy stain cells with
Hoechst 33342 (see Note 11):
(a) Aspirate medium from wells of multiwell plate.
(b) Add Hoechst 33342 solution at 1 μg/ml in PBS.
(c) Incubate for 10 min at RT (20–25
C).
(d) Rinse three times with PBS.
3.3 Preparation
of Vaccine Mixtures
1. Prepare plasmid DNA solution in PBS, pH 7.4 at required
concentration, (e.g., 2.0 μg/μl).
2. Prepare vaccine mixtures with reagent A consisting of PTAI-68/PTAI-7 + DOPE with DMEM F-12 Ham prepared in Subheading 3.1 (step 4(a)).
(a) Prepare one dose of vaccine containing 125 μg DNA in
final volume of 160 μl (PTAI–DNA ratio 0.8:1 w/w) as
follows:
– Mix 75 μl of prepared PTAI composition with 85 μl of
plasmid DNA solution containing 125 μg DNA (e.g.,
62.5 μl plasmid solution at concentration 2 μg/μ
l + 22.5 μl PBS, pH 7.4).
– Incubate for 30 min at RT (20–25
C) with gentle
rocking.
3. Prepare vaccine mixtures with reagent B consisting of PTAI10-14/PTAI-11 + DOPE + DC-chol with DMEM F-12 Ham
prepared in Subheading 3.1 (step 4(b)).
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Monika Rak et al.
– 93.3 μl of DMEM F-12 Ham (for DU145 cells) or
EMEM (for XC cells) and 106.7 μl of PTAI-1014/PTAI-11 based lipoplexes prepared in step 5
(b).
7. After adding lipoplexes, gently slide the plate in a cross-like
pattern (forward and backward and side-to-side) or in a figureeight-like pattern three to four times.
8. Incubate cells at 37
C with 5% CO 2 for 5 h.
9. Add 400 μl of DMEM F-12 Ham (for DU145 cells) or EMEM
(for XC cells) supplemented with 20% FCS or FBS and
200 IU/ml penicillin, 20 μg/ml neomycin, and 20 μg/ml
streptomycin.
10. Verify transfection efficiency 24 h after transfection (see Note
10) by fluorescence microscopy or flow cytometry. Examples
of PTAI efficiency as components of lipofecting mixtures used
in vitro are shown in Fig. 2b and c.
Optional: for fluorescent microscopy stain cells with
Hoechst 33342 (see Note 11):
(a) Aspirate medium from wells of multiwell plate.
(b) Add Hoechst 33342 solution at 1 μg/ml in PBS.
(c) Incubate for 10 min at RT (20–25
C).
(d) Rinse three times with PBS.
3.3 Preparation
of Vaccine Mixtures
1. Prepare plasmid DNA solution in PBS, pH 7.4 at required
concentration, (e.g., 2.0 μg/μl).
2. Prepare vaccine mixtures with reagent A consisting of PTAI-68/PTAI-7 + DOPE with DMEM F-12 Ham prepared in Subheading 3.1 (step 4(a)).
(a) Prepare one dose of vaccine containing 125 μg DNA in
final volume of 160 μl (PTAI–DNA ratio 0.8:1 w/w) as
follows:
– Mix 75 μl of prepared PTAI composition with 85 μl of
plasmid DNA solution containing 125 μg DNA (e.g.,
62.5 μl plasmid solution at concentration 2 μg/μ
l + 22.5 μl PBS, pH 7.4).
– Incubate for 30 min at RT (20–25
C) with gentle
rocking.
3. Prepare vaccine mixtures with reagent B consisting of PTAI10-14/PTAI-11 + DOPE + DC-chol with DMEM F-12 Ham
prepared in Subheading 3.1 (step 4(b)).
398
Monika Rak et al.