19. CaCl 2 50 mM: Dissolve 55.5 mg in 10 mL deionized water.
20. Digestion buffer: Mix 10 mL 1 M Tris, 4 mL 50 mM CaCl 2 ,
10 mL acetonitrile to a total volume of 100 mL.
21. Formic acid 10%: Mix 10 mL of formic acid and 90 mL HPLC
water.
22. C18 wash buffer: Mix 80 mL acetonitrile, 1 mL 10% Formic
acid and 19 mL HPLC water.
23. C18 loading buffer: Mix 2 mL acetonitrile, 1 mL 10% formic
acid, and 19 mL HPLC water.
24. C18 elution buffer: Combine 50 mL acetonitrile, 1 mL 10%
formic acid, and 49 mL HPLC water.
3 Methods
3.1 Transduction
Assay Using
FluorescenceActivated Cell Sorting
This protocol details how to perform an in vitro transduction assay
of a candidate CPP for the purposes of validation. Here we provide
a protocol to validate the specific transduction of Cy5.5-CTP into
H9C2 cells with 3T3 mouse fibroblasts as a negative control.
1. Thaw cells from À180
C liquid N 2 storage and plate onto a
T-75 tissue culture treated flask with vented caps. Passage
H9C2 cells for a minimum of three passages, trypsinizing at
60–70% confluence and plating at a 1:3 to 1:5 dilution (one
70% confluent T-75 flask yields ~one million cells) (see Note 1).
2. Passage the 3T3 cells for a minimum of 3 passages (similar to
details given above for H9C2 cells), after thawing from
À180
C (see Note 1).
3. Once cells are 70% confluent, aspirate media, wash once with
pre-warmed PBS, aspirate PBS and add 2 mL of Trypsin/
EDTA, incubate for 5–7 min until cells have rounded up and
are coming off in sheets.
4. Neutralize with 10–12 mL of pre-warmed media, collect cells,
spin at 300 Â g for 6 min to pellet the cells.
5. Aspirate supernatant and wash cell pellet twice with 10–12 mL
of pre-warmed media.
6. Count cells using a hematocytometer and plate at a density of
1 Â 10
5 cells/well of a 12-well plate.
7. Twenty-four hours post-plating, aspirate and replace with
pre-warmed 1 mL of media to which 10 μM of Cy5.5-CTP
final concentration and 1 μL/mL of Live/Dead Fixable Aqua
Dead Cell Stain has been added (see Notes 2 and 3). Return to
incubator for 30 min. All cell works/groups/treatments are
done in triplicate.
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Kyle S. Feldman et al.
20. Digestion buffer: Mix 10 mL 1 M Tris, 4 mL 50 mM CaCl 2 ,
10 mL acetonitrile to a total volume of 100 mL.
21. Formic acid 10%: Mix 10 mL of formic acid and 90 mL HPLC
water.
22. C18 wash buffer: Mix 80 mL acetonitrile, 1 mL 10% Formic
acid and 19 mL HPLC water.
23. C18 loading buffer: Mix 2 mL acetonitrile, 1 mL 10% formic
acid, and 19 mL HPLC water.
24. C18 elution buffer: Combine 50 mL acetonitrile, 1 mL 10%
formic acid, and 49 mL HPLC water.
3 Methods
3.1 Transduction
Assay Using
FluorescenceActivated Cell Sorting
This protocol details how to perform an in vitro transduction assay
of a candidate CPP for the purposes of validation. Here we provide
a protocol to validate the specific transduction of Cy5.5-CTP into
H9C2 cells with 3T3 mouse fibroblasts as a negative control.
1. Thaw cells from À180
C liquid N 2 storage and plate onto a
T-75 tissue culture treated flask with vented caps. Passage
H9C2 cells for a minimum of three passages, trypsinizing at
60–70% confluence and plating at a 1:3 to 1:5 dilution (one
70% confluent T-75 flask yields ~one million cells) (see Note 1).
2. Passage the 3T3 cells for a minimum of 3 passages (similar to
details given above for H9C2 cells), after thawing from
À180
C (see Note 1).
3. Once cells are 70% confluent, aspirate media, wash once with
pre-warmed PBS, aspirate PBS and add 2 mL of Trypsin/
EDTA, incubate for 5–7 min until cells have rounded up and
are coming off in sheets.
4. Neutralize with 10–12 mL of pre-warmed media, collect cells,
spin at 300 Â g for 6 min to pellet the cells.
5. Aspirate supernatant and wash cell pellet twice with 10–12 mL
of pre-warmed media.
6. Count cells using a hematocytometer and plate at a density of
1 Â 10
5 cells/well of a 12-well plate.
7. Twenty-four hours post-plating, aspirate and replace with
pre-warmed 1 mL of media to which 10 μM of Cy5.5-CTP
final concentration and 1 μL/mL of Live/Dead Fixable Aqua
Dead Cell Stain has been added (see Notes 2 and 3). Return to
incubator for 30 min. All cell works/groups/treatments are
done in triplicate.
104
Kyle S. Feldman et al.