Macrophage Uptake of CNS1-1D
Mouse Peritoneal Macrophages
• To see the uptake of the SPIO by the
• Mice were pre-treated with mineral oil
for 2 days to stimulate the proliferation
of peritoneal macrophages
• CNS-1-1d-FL/CNS-1-1d was injected
• 24 hours later peritoneal macrophages
were harvested by washing
• These macrophages were grown in
slide chambers or tissue culture flasks.
• 24 to 48 hours after culture, the cells
were fixed with formalin.
• Pictures were taken with white light
and UV light.
• Some cultures were saved for 2 weeks.
• Some of the slides were stained with
DAPI for nuclear staining.
• The fluorescence labeled SPIO are
visible inside the macrophages.
• Macrophages that had SPIO uptake
could be magnetically separated.
Our Study (Fluorometry)
• Using a 96-welled slide chamber we added 190μL
of PBS (phosphate buffer solution) to 6 wells.
• 10μL of Fl/Cold/Rhodamine SPIO were added to
• 100μl of this solution was added to the next row of
wells and mixed with the PBS already present in
• 100μl from this well was added to the next so that
the concentration of the SPIO decreased by 50%
in the subsequent wells.
• This procedure was followed till we reached a
concentration of 0% of the SPIO in the 11th
• We measured the Fluorometry of this solution
• We followed the same procedure on a macrophage
slide and incubated the macrophages with the
solution for 3 and 6 hrs.
Comparision of SPIO_FL content of
Macrophages after 3 and 6 hours Incubation
• The decrease in fluorometry could be due to
wash out of the macrophages from the slide
• Leakage from the macrophages due to cell