Hi everyone, I am trying to measure intracellular iron content from cultured cells using the colorimetry method (chromogen reagent), however so far unsuccessful. The absorbance readings at 540 nm of my samples are almost zero, while that of my standards (made from FeSO4) are fantastic (R= 0.999). Could the reason be on the way I prepare the samples? or is it just the concentration of iron in the sample too low to be detected by colorimteric method? This is how I prepare my sample:
Day 0- Cultured cells to confluency using medium + FCS
Day 2- Changed medium to serum free (- FCS) 24 h
Day 3- Revert to medium with serum before lysing. Wash with ice-cold PBS—> Trypsinize —> centrifuge —> wash with PBS (to remove trypsin)
- Added acid solution to the cell pellet —> incubated ON at 65 oC
Day 4- Cool and spin samples —> pipette into 96-well plate, (standards are included on the same plate)
- Add 200 µl chromogen reagent
- Measured absorbance at 540 nm wavelength
Any suggestions will be highly appreciated.
Hi,
I think that concentration of iron in cell culture is too low to be detected by this method. Colorimetry method using i.e. bathophenantroline sulfate (BPS) works well with animal tissues such as liver and spleen where iron content is high.
I would suggest you to use more sensitive fluorescent probes such as calcein-AM or RhoNox-1 to detect intracellular iron. You can use plate reader or (even better) flow cytometry for measurement.
Good luck and best regards,
Magda
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