9.5 Experimental Section
181
For fluorescence microscopy imaging, all the PDMS substrates were placed upside
down on the glass slides.
Immunofluorescent Staining for Actin Cytoskeleton and Focal Adhesion of
Captured Cells. Firstly, the cells captured on the substrates were rinsed once with
PBS buffer. Secondly, the cells were treated with 4% PFA and 0.1% Triton X-100 in
PBS buffer for 5 min at room temperature. Thirdly, the cells were covered with 4%
PFA in PBS buffer for 20 min at room temperature, followed by washing the cells
three times with PBS buffer. Fourthly, the cells were immersed into 50 mM ammonium chloride solution for 10 min for background reduction, followed by washing
the cells twice with PBS buffer. Fifthly, the cells were permeabilized with 0.2%
Triton X-100 in PBS buffer for 10 min at room temperature, followed by washing
the cells three times with PBS buffer. Sixthly, a blocking solution (2% BSA in PBS
buffer) was applied for 45 min at room temperature. The blocking solution was then
removed and the cells were not washed subsequently. Seventhly, primary antibody
(mouse anti-paxillin clone 349) diluted 1:100 in PBS buffer containing 2% BSA
was added for each substrate and left to incubate overnight at 4 °C. The cells were
then washed three times with PBS buffer. Eighthly, secondary antibody (goat antimouse lgG-Alexa488) diluted 1:500 in PBS buffer containing 2% BSA was added
for each substrate and left to incubate for 1 h at room temperature in the dark. Then,
it was washed three times with PBS buffer. Ninthly, Phalloidin-Rhodamin diluted
1:40 in PBS buffer containing 1% BSA was added to each substrate and incubated
for 30 min at room temperature. Then, this was washed three times with PBS buffer.
Tenthly, Hoechst 33,258 (1 μg ml
−1 ) was added for 15 min at room temperature,
followed by washing the cells three times with PBS buffer. The fluorescence images
were obtained by a fluorescence microscope (Axiovert 135, Carl Zeiss, Oberkochen,
Germany). For fluorescent imaging, all the PDMS substrates were placed upside
down on the glass slides.
Scanning Electron Microscopy (SEM). The morphologies of PaTu 8988t and
MCF7 cells captured on patterned and flat PDMS substrates were analyzed by
SEM. After incubation, cells were washed with PBS buffer twice and then fixed by
glutaraldehyde (3%(v/v) in PBS) for 30 min at room temperature. Subsequently, the
surfaces were washed twice with PBS buffer and twice with Milli-Q water. After that,
cells were dehydrated through a graded ethanol series (30, 50, 70, 90, and 100%). For
surface drying, the samples were immersed in hexamethydisiloxane (HMDS) three
times until there was only HMDS on the cells left. The last HMDS was evaporated
in a fume hood. Finally, the samples were sputter coated with gold and imaged in a
CamScan microscope (CS24, Applied Beams, Beaverton, USA).
Cell Release. To achieve cell release, the PDMS substrates (with captured cells)
were immersed into glutathione solution (10 mg ml
−1 in PBS). After 1 h incubation
at 37 °C, the cells were aspirated by gently pipetting and the cell suspension was
transferred to a centrifuge tube. After centrifuging (1200 rpm for 4 min, centrifuge
produced by Thermo Fisher Scientific, Germany), the released cells were added to
the wells of a new 24-well plate for the following analysis.
181
For fluorescence microscopy imaging, all the PDMS substrates were placed upside
down on the glass slides.
Immunofluorescent Staining for Actin Cytoskeleton and Focal Adhesion of
Captured Cells. Firstly, the cells captured on the substrates were rinsed once with
PBS buffer. Secondly, the cells were treated with 4% PFA and 0.1% Triton X-100 in
PBS buffer for 5 min at room temperature. Thirdly, the cells were covered with 4%
PFA in PBS buffer for 20 min at room temperature, followed by washing the cells
three times with PBS buffer. Fourthly, the cells were immersed into 50 mM ammonium chloride solution for 10 min for background reduction, followed by washing
the cells twice with PBS buffer. Fifthly, the cells were permeabilized with 0.2%
Triton X-100 in PBS buffer for 10 min at room temperature, followed by washing
the cells three times with PBS buffer. Sixthly, a blocking solution (2% BSA in PBS
buffer) was applied for 45 min at room temperature. The blocking solution was then
removed and the cells were not washed subsequently. Seventhly, primary antibody
(mouse anti-paxillin clone 349) diluted 1:100 in PBS buffer containing 2% BSA
was added for each substrate and left to incubate overnight at 4 °C. The cells were
then washed three times with PBS buffer. Eighthly, secondary antibody (goat antimouse lgG-Alexa488) diluted 1:500 in PBS buffer containing 2% BSA was added
for each substrate and left to incubate for 1 h at room temperature in the dark. Then,
it was washed three times with PBS buffer. Ninthly, Phalloidin-Rhodamin diluted
1:40 in PBS buffer containing 1% BSA was added to each substrate and incubated
for 30 min at room temperature. Then, this was washed three times with PBS buffer.
Tenthly, Hoechst 33,258 (1 μg ml
−1 ) was added for 15 min at room temperature,
followed by washing the cells three times with PBS buffer. The fluorescence images
were obtained by a fluorescence microscope (Axiovert 135, Carl Zeiss, Oberkochen,
Germany). For fluorescent imaging, all the PDMS substrates were placed upside
down on the glass slides.
Scanning Electron Microscopy (SEM). The morphologies of PaTu 8988t and
MCF7 cells captured on patterned and flat PDMS substrates were analyzed by
SEM. After incubation, cells were washed with PBS buffer twice and then fixed by
glutaraldehyde (3%(v/v) in PBS) for 30 min at room temperature. Subsequently, the
surfaces were washed twice with PBS buffer and twice with Milli-Q water. After that,
cells were dehydrated through a graded ethanol series (30, 50, 70, 90, and 100%). For
surface drying, the samples were immersed in hexamethydisiloxane (HMDS) three
times until there was only HMDS on the cells left. The last HMDS was evaporated
in a fume hood. Finally, the samples were sputter coated with gold and imaged in a
CamScan microscope (CS24, Applied Beams, Beaverton, USA).
Cell Release. To achieve cell release, the PDMS substrates (with captured cells)
were immersed into glutathione solution (10 mg ml
−1 in PBS). After 1 h incubation
at 37 °C, the cells were aspirated by gently pipetting and the cell suspension was
transferred to a centrifuge tube. After centrifuging (1200 rpm for 4 min, centrifuge
produced by Thermo Fisher Scientific, Germany), the released cells were added to
the wells of a new 24-well plate for the following analysis.