To begin, use a multi-channel pipette to distribute 50 microliters of PBS into columns 2 to 10 of rows A to E on a 96-well plate. Pipette 100 microliters of two millimolar ferric ammonium sulfate stock solution into column 11. With a multi-channel pipette, transfer 50 microliters of the ferric ammonium sulfate stock from column 11 into column 10.
Pipette the solutions multiple times to mix well. Perform step-wise double dilution across the columns from 9 to 2 and row A to 2. Now pipette out 50 microliters of the solution from column 2 to discard.
After adding PBS into all the wells, pipette 10 microliters of 10 micromolar Calcein stock into each well. For the standalone PBS control, pipette 100 microliters of PBS into row F of columns 1 to 5. Then add 100 microliters of two millimolar ferric ammonium sulfate to row F of columns 6 to 10 for the FAS control.
Incubate the plate at room temperature in the dark for 10 minutes. Analyze the fluorescence with a microplate reader. Pipette 50 microliters of PBS to columns 2 to 10 of rows A to E of a 96-well plate.
To column 11, add 100 microliters of two micromolar stock solution of the iron chelator solution. With a multichannel manual pipette, remove 50 microliters of the iron chelator solution from column 11. Then transfer it into the PBS in column 10 from rows A to E.Pipette up and down to mix the solution well.
Continue to dilute the chelator solution across the remaining columns. Discard 50 microliters of the solution from the wells of column 2, then pipette 30 microliters of PBS into all wells. Using a multichannel pipette, add 10 microliters of 100 micromolar ferric ammonium sulfate stock into columns 2 to 12 of rows A to E.Add 10 microliters of 10 micromolar Calcein stock into the same wells.
Next for the positive control, pipette PBS, Calcein stock, and ferric ammonium sulfate stock into column 1 of rows A to E.Mix the samples well by pipetting the solutions up and down. Then add 100 microliters of PBS into row F of columns 1 to 5 to create the PBS negative control. Pipette the same volume of the iron chelator solution to row F from column 6 to 10 to create the standalone chelator control.
Place the well plate at room temperature for 10 minutes in the dark. Then analyze on a multimodal microplate reader as before. The addition of ferrous ions to Calcein resulted in a linear decrease in Calcein fluorescence.
The iron chelator outcompeted Calcein for the ferrous ions. The chelator increased the Calcein fluorescence up to a peak.
