FLIPR Complete Calibration with known kP ( IAFLIPRCompleteKP )

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.
K+ Steps Ranges	KStepsRanges	string	Enter a list of range definitions for the duration of the K+-steps (in the above defined time units), including the MDC treatment.
[K+]ec Steps (mM;mM)	KStepsConcentrations	string	Extracellular (ec) K+ concentrations corresponding to the above defined steps, including MDC, in mM. Use the Wizard tab to calculate concentrations from added volumes.
kP	kP	real	Previously measured value of kp
CDC Range	CDCRange	string	The range of Complete Depolarization Cocktail addition (in the above defined time units).
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.
PN (permeability of plasma membrane to ions other than K+)	PN	real	Summed relative permeability of the plasma membrane to ions other than K+ during * concentration, shown in the numerator of the GHK equation. Use -1 for automatic determination.
fP0-fPX Allowed Range	fP0MinusfPXRange	string	Trace selection criterion: the minimum and maximum allowed value of fP0-fPX
fPX r^2 Min	fpXr2Min	real	Trace selection criterion: the minimum allowed value of r^2 for the FLIPR-regression.
Quality control by propagated error of baseline	QCPMPrest	boolean	Removes traces where the estimated SE of baseline is greater than the threshold below. Requires error propagation.
Limit SE of baseline (mV)	QCPMPrestMaxSE	real	Traces where the SE of resting ΔψP parameter is larger than this number are removed.

Description:

This method calculates plasma membrane potential (ΔψP) time course by calibration parameters from a single time lapse with the exception of kP using the FLIPR Membrane Potential Kit from Molecular Devices (aka PMPI, plasma membrane potential indicator). The redistribution of FLIPR is relatively fast and the exact value of kP has little effect unless very fast oscillations are looked. Therefore, in many cases the kP=0.38 determined on hippocampal neurons can be used.
Required experimental protocol: any time lapse, followed by establishment of K-equilibrium potential, at least one addition of high K+-medium and finally complete depolarization. Typically use 3-4 steps.
The cells have to be able to maintain a constant hyperpolarized ΔψP before starting K+-steps. Do not use this method if ΔψP depolarizes substantially when adding MDC.