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Macrophage uptake of cns1 1 d ( in vitro study)


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Macrophage uptake of cns1 1 d ( in vitro study)

  1. 1. Macrophage Uptake of CNS1-1D (our SPIO) In Mouse Peritoneal Macrophages Done by Dr. Chan
  2. 2. Objectives • To see the uptake of the SPIO by the macrophages.
  3. 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. 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. 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. 6. Macrophage Uptake of Fl-CNS
  7. 7. …..Continued (with DAPI Staining)
  8. 8. ……Continued
  9. 9. Results • The fluorescence labeled SPIO are visible inside the macrophages. • Macrophages that had SPIO uptake could be magnetically separated.
  10. 10. 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.
  11. 11. …..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.
  12. 12. 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
  13. 13. 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