3 Methods
3.1 Preparation
of Lipid-Based Vaccine
Carriers
1. Dissolve PTAI-6-8 (or PTAI-7), PTAI-10-14 (or PTAI-11),
DOPE, and DC-cholesterol in ethanol (99%) to achieve concentrations: 20, 10, 25, and 33.3 mg/ml, respectively (see
Note 4).
2. To prepare 1 ml of reagent in a 2 ml glass vial mix:
(a) 85.80 μl PTAI-6-8 (or PTAI-7) and 51.32 μl DOPE (for
reagent A consisting of PTAI-6-8/PTAI-7 + DOPE)—
molar ratio of PTAI–DOPE 1.5:1.
(b) 166.86 μl PTAI-10-14 (or PTAI-11) with 53.14 μl
DOPE and 28.76 μl DC-cholesterol (for reagent B consisting of PTAI-10-14/PTAI-11 + DOPE + DC-chol)—
molar ratio of PTAI–DOPE–DC-chol 1:1:1.
3. Vortex at the highest vortex settings for 1 min at RT
(20–25
C).
4. Add ethanolic solution of lipids to DMEM F-12 Ham medium
(see Note 5).
(a) 114.26 μl of lipids to 885.74 μl of medium for reagent A.
(b) 207.30 μl of lipids to 792.70 μl of medium for reagent B.
5. Vortex at the highest vortex settings for 3 min at RT
(20–25
C).
6. Use immediately or store reagents at 4
C (see Note 6).
3.2 Carriers Activity
Testing In Vitro
1. Prepare culture media:
(a) DMEM F-12 Ham (for DU145 cells) or/and EMEM (for
XC cells) supplemented with 10% FCS or FBS.
(b) DMEM F-12 Ham (for DU145 cells) or/and EMEM (for
XC cells) supplemented with 20% FCS or FBS and
200 IU/ml penicillin, 20 μg/ml neomycin, and 20 μg/
ml streptomycin.
2. Seed DU145 or XC cells into the wells of a 24-well plate at a
density of 8 Â 10
4 cell/well in 500 μl of DMEM F-12 Ham
(for DU145 cells) or/and EMEM (for XC cells) supplemented
with 10% FCS or FBS without antibiotics (see Note 7).
3. Cultivate cells in a humidified atmosphere with 5% CO 2 at
37
C for 24 h (to reach 70–80% confluence).
4. After 24 h, on the day of testing, prepare the PTAI-based
reagents as described in Subheading 3.1 or take them out of
the 4
C storage conditions in advance. When they reach RT
(20–25
C), they are ready to be used. Vortex the reagents at
the highest vortex settings for 3 min at RT (20–25
C) immediately before use.
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Monika Rak et al.
3.1 Preparation
of Lipid-Based Vaccine
Carriers
1. Dissolve PTAI-6-8 (or PTAI-7), PTAI-10-14 (or PTAI-11),
DOPE, and DC-cholesterol in ethanol (99%) to achieve concentrations: 20, 10, 25, and 33.3 mg/ml, respectively (see
Note 4).
2. To prepare 1 ml of reagent in a 2 ml glass vial mix:
(a) 85.80 μl PTAI-6-8 (or PTAI-7) and 51.32 μl DOPE (for
reagent A consisting of PTAI-6-8/PTAI-7 + DOPE)—
molar ratio of PTAI–DOPE 1.5:1.
(b) 166.86 μl PTAI-10-14 (or PTAI-11) with 53.14 μl
DOPE and 28.76 μl DC-cholesterol (for reagent B consisting of PTAI-10-14/PTAI-11 + DOPE + DC-chol)—
molar ratio of PTAI–DOPE–DC-chol 1:1:1.
3. Vortex at the highest vortex settings for 1 min at RT
(20–25
C).
4. Add ethanolic solution of lipids to DMEM F-12 Ham medium
(see Note 5).
(a) 114.26 μl of lipids to 885.74 μl of medium for reagent A.
(b) 207.30 μl of lipids to 792.70 μl of medium for reagent B.
5. Vortex at the highest vortex settings for 3 min at RT
(20–25
C).
6. Use immediately or store reagents at 4
C (see Note 6).
3.2 Carriers Activity
Testing In Vitro
1. Prepare culture media:
(a) DMEM F-12 Ham (for DU145 cells) or/and EMEM (for
XC cells) supplemented with 10% FCS or FBS.
(b) DMEM F-12 Ham (for DU145 cells) or/and EMEM (for
XC cells) supplemented with 20% FCS or FBS and
200 IU/ml penicillin, 20 μg/ml neomycin, and 20 μg/
ml streptomycin.
2. Seed DU145 or XC cells into the wells of a 24-well plate at a
density of 8 Â 10
4 cell/well in 500 μl of DMEM F-12 Ham
(for DU145 cells) or/and EMEM (for XC cells) supplemented
with 10% FCS or FBS without antibiotics (see Note 7).
3. Cultivate cells in a humidified atmosphere with 5% CO 2 at
37
C for 24 h (to reach 70–80% confluence).
4. After 24 h, on the day of testing, prepare the PTAI-based
reagents as described in Subheading 3.1 or take them out of
the 4
C storage conditions in advance. When they reach RT
(20–25
C), they are ready to be used. Vortex the reagents at
the highest vortex settings for 3 min at RT (20–25
C) immediately before use.
396
Monika Rak et al.