HEY and MCF7 cells were seeded in 6-well plates at a density of 5.0 × 105 cells/well and grown overnight, and treated at the concentration indicated for 24 h. Cells were loaded with 0.25 μM calcein acetoxymethyl ester (CA-AM) (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37 °C, then washed with PBS1X and treated or not with 200 mM 3-hydroxy-1,2-dimethyl-4(1H)-pyridone (deferiprone or L1) (Sigma-Aldrich, St. Louis, MO, USA). Following staining, and washing with PBS, cells were analyzed using a FACS BD LSRFortessaTM X-20 cytofluorometer (BD Biosciences). The ∆ mean fluorescence intensity (∆MFI) between chelator-treated and untreated cells reflected the amount of LIP [54 (link)].
Calcein acetoxymethyl ester ca am
Manufactured by Merck Group
Sourced in United States
Calcein acetoxymethyl ester (CA-AM) is a fluorescent dye used in cell biology and biochemistry applications. It is a cell-permeant indicator that can be used to measure intracellular calcium levels. CA-AM is non-fluorescent until the acetoxymethyl ester group is cleaved by intracellular esterases, upon which it becomes fluorescent.
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2 protocols using calcein acetoxymethyl ester ca am
1
Cellular Labile Iron Pool Measurement
2
Quantification of Labile Iron Pool
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To quantify LIP, 0.5 × 106 cells were collected and washed with PBS. Then cells were incubated with 0,5 μM calcein acetoxymethyl ester (CA-AM) (Sigma- Aldrich) for 15 min at 37 °C. After cell washing, samples were incubated with a high-affinity chelator, 100 μM deferiprone (DF) (Sigma- Aldrich), at 37 °C for 1 h. Cells were washed 3 times in phosphate-buffered saline (PBS) at 1500 rpm for 5 min and then analyzed by flow cytometry (MACSQuant Analyzer 10, Miltenyi Biotec). The difference in the MFI before and after treatment with DF was used to calculate the amount of LIP (ΔF = MFICA-AM/DF-MFICA-AM).
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