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Macrophage Uptake of CNS1-1D
(our SPIO)
In
Mouse Peritoneal Macrophages
Done by
Dr. Chan
Objectives
• To see the uptake of the SPIO by the
macrophages.
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
…Continued
• These macrophages were grown in
slide chambers or tissue culture flasks.
• 24 to 48 hours after culture, the cells
were fixed with formalin.
…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.
Macrophage Uptake of Fl-CNS
…..Continued (with DAPI
Staining)
……Continued
112 macrophage uptake of cns1 1 d
112 macrophage uptake of cns1 1 d
112 macrophage uptake of cns1 1 d
Results
• 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
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.
…..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.
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
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

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112 macrophage uptake of cns1 1 d

  • 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.
  • 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