FLIPR Complete Iterative Calibration ( IAFLIPRIterative )

Parameters:

Name	Short Name	Type	Description
Range definitions in time courses	rangedef	string	Defines whether the ranges given below refer to frames or seconds in the recordings
Baseline Range	BaselineRange	string	Defines the range of the baseline (in the above defined time units). Longer baseline results more accurate calibration.
CDC Range	CDCRange	string	The range of Complete Depolarization Cocktail addition (in the above defined time units).
kP	kP	real	Previously measured value of kp
fP0-fPX Allowed Range	fP0MinusfPXRange	string	Trace selection criterion: the minimum and maximum allowed value of fP0-fPX
Take maximum of CDC Range	CDCMax	boolean	If set yes, the maximum FLIPR intensity within the CDC Range is taken as zero calibration value (it error is the local SD of the range). Otherwise mean is calculated.
Part complete ΔψP depolarization range	PartCDCRange	string	Defines the range after CDC addition, where some small ΔψP is maintained by Donnan equilibrium.
ΔψP at baseline is	PMPway	string	Defines whether baseline ΔψP is calculated and used indepenedntly for each trace or averaged and uniformly used for all traces. In the latter case baseline ΔψP is calculated for each trace, and then the calibration is re-run using an average baseline ΔψP value.

Description:

This method calculates plasma membrane potential (ΔψP) time course with the help of TMRM data without apriori knowledge of baseline ΔψP or PN, only assuming the value of kP using the FLIPR Membrane Potential Kit from Molecular Devices (aka PMPI, plasma membrane potential indicator).
Works only in combination with the Complete or Complete (known k) ΔψM calibration. The algorithm exploits the feature of TMRM calibration that at partial ΔψP deploarization and complete mitochondrial depolarization an inaccurate baseline ΔψP causes non-zero calculated ΔψM. baseline ΔψP is determined by minimization of calibrated ΔψM at part complete ΔψP depolarization. Required experimental protocol: baseline, followed by any time lapse, then MDC and CDC.
Experiments where CDC (PFA) causes gradual depolarization of ΔψP are amenable to this method.
Select a range at the beginning of CDC (PFA) application as "Part. Depol CDC", where the FLIPR fluorescence has not yet reached a plateau.
Select "CDC (Zero)" where the FLIPR signal has reached a plateau.
Important: to work with this algorithm, spectral unmixing need to be precisely set up. It is not recommended turning on calculation of residual bleedthrough calculation in the "Constants" section of the wizard because it may introduce large errors.
Also set up "Complete" ΔψM calibration as given below: