8. Suggested groups are H9C2/3T3 cells with no treatment or
random peptide or CTP with Live/Dead stain added to all
groups.
9. After the incubation period, wash cells extensively (at least 3Â)
with pre-warmed PBS, trypsinize, and centrifuge to collect cell
pellet.
10. Wash the cell pellet with media once.
11. Aspirate the supernatant media above the cell pellet and resuspend in 1 mL of FACS Fixation Buffer.
12. Incubate the cells for 10 min at room temperature.
13. Add up to 5 mL of PBS and centrifuge to collect cell pellet.
14. Aspirate the supernatant and resuspend the cells in 200 μL
of PBS.
15. Store the resuspended cells on ice, light protected.
16. In the flow cytometer run bleach and distilled water for 1 min
each to clear the lines. Set to standby with a tube of distilled
water.
17. Set up a protocol, specifying number of events to log and gates.
Gates to set up would be to select for cells using
FSC-A vs. SSC-A graph, a gate to select for live cells excluding
the Live/Dead Fixable Aqua Dead Cell Stain using an
SSC-A vs. 405 nm violet laser 535/50 filter, and graph to
display SSC-A vs. 640 nm red laser 660/20 filter.
18. Vortex the cell sample, insert into the cytometer and adjust
voltages so that the 660/20 fluorescence peaks in the middle of
the graph. Apply these settings to the rest of the samples.
3.2 Transduction
Assay Using Confocal
Microscopy
This protocol is to acquire qualitative data about the transduction
of a CPP using confocal microscopy. Here we utilize H9C2 cells
incubated with Cy5.5 labeled CTP with 3T3 cells as a negative
control. This protocol can be modified for use in ther cell lines,
peptides, and fluorophores and can be modified for simultaneous
fluorescent labeling of different cellular organelles for co-localization of CPP to particular cellular compartments.
1. Passage H9C2 cells and 3T3 cells as detailed in Subheading 3.1
above and plate in an optical glass-bottomed dish at a cell
density of 5 Â 10
4 cells/well.
2. Twenty-four hours post-plating, aspirate, and replace with
pre-warmed media to which Cy5.5-CTP has been added to
give a final concentration of 10 μM.
3. Separate group of cells should be treated with vehicle (PBS)
only and a random peptide similarly labeled with Cy5.5.
Validating Cardiac Targeting Peptide
105
random peptide or CTP with Live/Dead stain added to all
groups.
9. After the incubation period, wash cells extensively (at least 3Â)
with pre-warmed PBS, trypsinize, and centrifuge to collect cell
pellet.
10. Wash the cell pellet with media once.
11. Aspirate the supernatant media above the cell pellet and resuspend in 1 mL of FACS Fixation Buffer.
12. Incubate the cells for 10 min at room temperature.
13. Add up to 5 mL of PBS and centrifuge to collect cell pellet.
14. Aspirate the supernatant and resuspend the cells in 200 μL
of PBS.
15. Store the resuspended cells on ice, light protected.
16. In the flow cytometer run bleach and distilled water for 1 min
each to clear the lines. Set to standby with a tube of distilled
water.
17. Set up a protocol, specifying number of events to log and gates.
Gates to set up would be to select for cells using
FSC-A vs. SSC-A graph, a gate to select for live cells excluding
the Live/Dead Fixable Aqua Dead Cell Stain using an
SSC-A vs. 405 nm violet laser 535/50 filter, and graph to
display SSC-A vs. 640 nm red laser 660/20 filter.
18. Vortex the cell sample, insert into the cytometer and adjust
voltages so that the 660/20 fluorescence peaks in the middle of
the graph. Apply these settings to the rest of the samples.
3.2 Transduction
Assay Using Confocal
Microscopy
This protocol is to acquire qualitative data about the transduction
of a CPP using confocal microscopy. Here we utilize H9C2 cells
incubated with Cy5.5 labeled CTP with 3T3 cells as a negative
control. This protocol can be modified for use in ther cell lines,
peptides, and fluorophores and can be modified for simultaneous
fluorescent labeling of different cellular organelles for co-localization of CPP to particular cellular compartments.
1. Passage H9C2 cells and 3T3 cells as detailed in Subheading 3.1
above and plate in an optical glass-bottomed dish at a cell
density of 5 Â 10
4 cells/well.
2. Twenty-four hours post-plating, aspirate, and replace with
pre-warmed media to which Cy5.5-CTP has been added to
give a final concentration of 10 μM.
3. Separate group of cells should be treated with vehicle (PBS)
only and a random peptide similarly labeled with Cy5.5.
Validating Cardiac Targeting Peptide
105