3.4 Methods and Materials
93
pre-incubation of cells at 4°C followed by 2 hours incubation with fluoresceinated
peptides at 4°C to assess the temperature dependence of fluorescent labeling.
2. Cell Cycle
For the flow cytometry of cell-cycle arrest experiments, cells were treated with
peptides and Nutlin-3a for 48 hours. Subsequently, cells were washed twice with
phosphate-buffered saline (PBS) and harvested by trypsinization. The cells were
fixed with cold 70% ethanol for 4 hours and then underwent centrifugation at
2000 rpm for 5 min to remove the ethanol. Then the fixed cells were dispersed in
PBS with 1% Triton-100, 1 mg/mL RNase and 5 mg/mL PI, stained at 37°C for
30 min (double fixation was not needed for this experiment). The samples were
analyzed using a FACS Calibur flow cytometer (Becton Dickinson, Mississauga,
CA). The percentages of cells in G1, S, and G2/M phases were determined by
FlowJo software.
3. Apoptosis assay
The apoptosis assay was conducted using the FITC Annexin-V/PI Apoptosis
Detection Kit I (BD Pharmingen TM) according to the manufacturer’s instructions. Approximately 1 × 10
6 cells were seeded in a six-well plate. Then, the
cells were treated with the peptides and nutlin-3a and incubated for 48 hours.
The cells were then collected and washed twice with cold PBS and suspended in
binding buffer. The induction of the apoptosis process enables the FITC-labeled
Annexin-V to bind with phosphatidylserine (PS) since it appears on the outer
surface of the cell membrane at the onset of apoptosis, and is confined to the
inner boundary of the cell membrane in healthy cells. The cell nuclei were stained
with propidium iodide (PI). The stained cells were analyzed by flow cytometry
to distinguish the apoptotic cells. The cells with positive florescent intensity
signals for both FITC and PI were representative of the apoptotic cell count. The
extent of apoptosis was measured through the detection of caspase-3 activity by
exposing the cells to a caspase-3-specific substrate (Oncogene). Fluorescence
as a result of substrate cleavage was measured in a Spectramax M5 microplate
reader (Molecular Devices).
3.4.5 Confocal Microscopy and Co-localization Assay
PA-1 cells (or MCF-7 cells) were cultured with DMEM with 10% FBS (v/v) in
imaging dishes (50000 cells/well) in a 37°C, 5% CO 2 incubator for one day until
they were about 80% adherent. Peptide was first dissolved in DMSO to make a
1 mM stock and then added to cells to a final concentration of 5 μM. The cells were
incubated with peptides for 1 hours at 37°C. After incubation, cells were washed 3
times with PBS and then fixed with 4% formaldehyde (Alfa Aesar, MA) in PBS for
10 minutes. They were then washed 3 times with PBS and stained with 1 μg/ml 4’,
6-diamidino-2-phenylindole (DAPI) (Invitrogen, CA) in PBS for 5 minutes. Images
of peptide localization in cells were taken via PerkinElmer confocal microscopy.
Image processing was done using the Volocity software package (Zeiss Imaging).
93
pre-incubation of cells at 4°C followed by 2 hours incubation with fluoresceinated
peptides at 4°C to assess the temperature dependence of fluorescent labeling.
2. Cell Cycle
For the flow cytometry of cell-cycle arrest experiments, cells were treated with
peptides and Nutlin-3a for 48 hours. Subsequently, cells were washed twice with
phosphate-buffered saline (PBS) and harvested by trypsinization. The cells were
fixed with cold 70% ethanol for 4 hours and then underwent centrifugation at
2000 rpm for 5 min to remove the ethanol. Then the fixed cells were dispersed in
PBS with 1% Triton-100, 1 mg/mL RNase and 5 mg/mL PI, stained at 37°C for
30 min (double fixation was not needed for this experiment). The samples were
analyzed using a FACS Calibur flow cytometer (Becton Dickinson, Mississauga,
CA). The percentages of cells in G1, S, and G2/M phases were determined by
FlowJo software.
3. Apoptosis assay
The apoptosis assay was conducted using the FITC Annexin-V/PI Apoptosis
Detection Kit I (BD Pharmingen TM) according to the manufacturer’s instructions. Approximately 1 × 10
6 cells were seeded in a six-well plate. Then, the
cells were treated with the peptides and nutlin-3a and incubated for 48 hours.
The cells were then collected and washed twice with cold PBS and suspended in
binding buffer. The induction of the apoptosis process enables the FITC-labeled
Annexin-V to bind with phosphatidylserine (PS) since it appears on the outer
surface of the cell membrane at the onset of apoptosis, and is confined to the
inner boundary of the cell membrane in healthy cells. The cell nuclei were stained
with propidium iodide (PI). The stained cells were analyzed by flow cytometry
to distinguish the apoptotic cells. The cells with positive florescent intensity
signals for both FITC and PI were representative of the apoptotic cell count. The
extent of apoptosis was measured through the detection of caspase-3 activity by
exposing the cells to a caspase-3-specific substrate (Oncogene). Fluorescence
as a result of substrate cleavage was measured in a Spectramax M5 microplate
reader (Molecular Devices).
3.4.5 Confocal Microscopy and Co-localization Assay
PA-1 cells (or MCF-7 cells) were cultured with DMEM with 10% FBS (v/v) in
imaging dishes (50000 cells/well) in a 37°C, 5% CO 2 incubator for one day until
they were about 80% adherent. Peptide was first dissolved in DMSO to make a
1 mM stock and then added to cells to a final concentration of 5 μM. The cells were
incubated with peptides for 1 hours at 37°C. After incubation, cells were washed 3
times with PBS and then fixed with 4% formaldehyde (Alfa Aesar, MA) in PBS for
10 minutes. They were then washed 3 times with PBS and stained with 1 μg/ml 4’,
6-diamidino-2-phenylindole (DAPI) (Invitrogen, CA) in PBS for 5 minutes. Images
of peptide localization in cells were taken via PerkinElmer confocal microscopy.
Image processing was done using the Volocity software package (Zeiss Imaging).