Macrophage Uptake of CNS1-1D (our SPIO)

InMouse Peritoneal Macrophages

Done byDr. 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

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

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0.3

0.4

0.5

0.6

0.7

100% 0.00%

3 hours6 hours

Fluorescence U

nit

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