Macrophage Uptake of CNS1-1D (our SPIO)

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Macrophage Uptake of CNS1-1D (our SPIO)

• In
• Mouse Peritoneal Macrophages
• Done by
• Dr. Chan

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Objectives

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

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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

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Continued

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

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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.

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Macrophage Uptake of Fl-CNS
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..Continued (with DAPI Staining)
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Continued
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Results

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

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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.

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..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.

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Comparision of SPIO_FL content of Macrophages
after 3 and 6 hours Incubation
Fluorescence Unit
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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