Im trying to assess if a physiological concentrations of insulin alters the phagocytic capacity of THP-1 cells in a hyperglycemic condition. Until now, our experiments showed that a hyperglycemic medium impairs the phagocytic capacity when these cells are challenged with L. amazonensis, now we wanna investigate if the addition of insulin alters this result, but we don't find what is the best insulin concentration in mol.
ITS (insulin/transferrin/selenite) supplements normally contain 1.0 mg/mL insulin and are available as 100x stocks (e.g. from Sigma/Merck: https://www.sigmaaldrich.com/catalog/product/sigma/i3146?lang=de®ion=DE or Gibco/Thermo Fisher: https://www.thermofisher.com/de/de/home/technical-resources/media-formulation.334.html). Dilution (1:100) of the supplement solution will give you a final insulin concentration of 10 µg/mL, equal to 0.1722 mmol/L (mM; M = 5,808 g/mol). 10 µg/mL is also the final insulin concentration I am working with in my experiments (purchased from Sigma: https://www.sigmaaldrich.com/catalog/product/sigma/i9278?lang=de®ion=DE)
Good luck - CL
Hi,
Same here with the ITS 100x supplement we add to the culture medium of our kidney cells (we use the one from thermo fisher). You can also consider a dose response approach since in vitro culture is only a partial reflect of the in vivo anyway especially if you expect an insulin signaling response in your cells (i'm not in the field, I don't know if immune cells do and whether it's part of your hypothesis). If THP cells happen to respond to supraphysiological doses, it is an in vitro model and it doesn't mean that wt untransformed monocytes would not at circulating concentrations in the human blood, which you could maybe verify on a relevant blood sample. And if you observe a restoration of phagocytosis along with insulin concentration increase it's probably good news.
Hope this helps
good luck
D_
I fixed these cells for 15' at 37 degrees with 3% - 4% (presumably) PFA.
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