On the basis of
our previous studies, a ratio of 100:1 for effector (p98 T cells)
to target (MMC cells) was selected for this study.25 (link),26 For fluorescence imaging, p98 T cells were labeled with Green Cell
Tracker dye (Invitrogen) according to the manufacturer’s protocol.
After 72 h of MMC cell culture in uncoated, Matrigel-precoated, and
PCgel-precoated 24-well TCPs, 106 p98 T cells (Green Cell
Tracker-labeled) were added. After 30 h of treatment, dead p98 T cells
were washed away from MMC cells using PBS. Dead MMC cells were stained
with SYTOX Blue nucleic acid stain (Invitrogen) according to the manufacturer’s
instructions. Cells were imaged using an inverted fluorescence microscope
(Nikon TE 300, Japan). To quantify the dead MMC cells after the treatment
with labeled p98 T cells, the signals from the images were quantified
through ImageJ. The dead cell percentage was calculated from the following
equation:
For SEM analysis, samples
were fixed with a 4% formaldehyde aqueous solution for 30 min at room
temperature. After the fixation and dehydration in a series of ethanol
washes (70, 85, 95, and 100%), the samples were dried with a critical
point dryer (Denton DCP-1, Cherry Hill, NJ). The samples were mounted
on SEM pin stub, sputter-coated with platinum, and then imaged with
a JSM-7000F SEM (JEOL, Tokyo, Japan).
our previous studies, a ratio of 100:1 for effector (p98 T cells)
to target (MMC cells) was selected for this study.25 (link),26 For fluorescence imaging, p98 T cells were labeled with Green Cell
Tracker dye (Invitrogen) according to the manufacturer’s protocol.
After 72 h of MMC cell culture in uncoated, Matrigel-precoated, and
PCgel-precoated 24-well TCPs, 106 p98 T cells (Green Cell
Tracker-labeled) were added. After 30 h of treatment, dead p98 T cells
were washed away from MMC cells using PBS. Dead MMC cells were stained
with SYTOX Blue nucleic acid stain (Invitrogen) according to the manufacturer’s
instructions. Cells were imaged using an inverted fluorescence microscope
(Nikon TE 300, Japan). To quantify the dead MMC cells after the treatment
with labeled p98 T cells, the signals from the images were quantified
through ImageJ. The dead cell percentage was calculated from the following
equation:
For SEM analysis, samples
were fixed with a 4% formaldehyde aqueous solution for 30 min at room
temperature. After the fixation and dehydration in a series of ethanol
washes (70, 85, 95, and 100%), the samples were dried with a critical
point dryer (Denton DCP-1, Cherry Hill, NJ). The samples were mounted
on SEM pin stub, sputter-coated with platinum, and then imaged with
a JSM-7000F SEM (JEOL, Tokyo, Japan).
