This study examined the uptake of a superparamagnetic iron oxide (SPIO) particle called CNS1-1D by mouse peritoneal macrophages. Mice were pre-treated to stimulate macrophage proliferation. CNS1-1D was injected into the peritoneum and macrophages were harvested 24 hours later. The macrophages were cultured and observed under light and UV light microscopy over 24-48 hours. Some macrophages that had taken up SPIO could be separated magnetically. A fluorometry study also showed decreasing SPIO fluorescence in macrophages over a concentration gradient and over 3-6 hours of incubation, possibly due to wash out or cell wall damage.

1. Macrophage Uptake of CNS1-1D
(our SPIO)
In
Mouse Peritoneal Macrophages
Done by
Dr. Chan

2. Objectives
• To see the uptake of the SPIO by the
macrophages.

3. Method
• 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
intraperitoneally.
• 24 hours later peritoneal macrophages
were harvested by washing

4. …Continued
• These macrophages were grown in
slide chambers or tissue culture flasks.
• 24 to 48 hours after culture, the cells
were fixed with formalin.

5. …Continued
• 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.

6. Macrophage Uptake of Fl-CNS

7. …..Continued (with DAPI
Staining)

8. ……Continued

12. Results
• The fluorescence labeled SPIO are
visible inside the macrophages.
• Macrophages that had SPIO uptake
could be magnetically separated.

13. 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
these chambers.
• 100μl of this solution was added to the next row of
wells and mixed with the PBS already present in
the wells.
• 100μl from this well was added to the next so that
the concentration of the SPIO decreased by 50%
in the subsequent wells.

14. …..Continued
• This procedure was followed till we reached a
concentration of 0% of the SPIO in the 11th
row of
wells.
• We measured the Fluorometry of this solution
with Fluoroscan.
• We followed the same procedure on a macrophage
slide and incubated the macrophages with the
solution for 3 and 6 hrs.

15. Comparision of SPIO_FL content of
Macrophages after 3 and 6 hours Incubation
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
100% 0.00%
3 hours
6 hours
FluorescenceUnit

16. Conclusion
• The decrease in fluorometry could be due to
wash out of the macrophages from the slide
surface
Or
• Leakage from the macrophages due to cell
wall damage