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FLIPR Calibration with [K+]ec steps and known [K+]ic and known kP
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FLIPR Complete Calibration with known kP
FLIPR Complete Calibration with known kP - Goldman
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ΔF/F0

FLIPR Complete Calibration with known kP - Goldman ( IAFLIPRCompleteKPGoldman )

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.
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.
Maximum SE of baseline (mV)	QCPMPrestMaxSE	real	Traces where the SE of resting ΔψP parameter is larger than this number are removed.
PN	PNway	string	Defines whether PN is calculated and used indepenedntly for each trace or a median value is used uniformly for all traces. In the latter case PN is calculated for each trace, and then the calibration is re-run using the median of the PN values for increased robustness. Note that errors in determination of PN are not propagated.
Weights for fitting K-steps	PNweights	string	Defines weighing least squares during nonlinear fit for colaculation of fpx and PN.

Description:

This method calculates plasma membrane potential (ΔψP) time course by calibration parameters from a single time lapse with only assuming the value of kP, using a nonlinear fit for Goldman equation to calculate K+-equilibrium potentials using the FLIPR Membrane Potential Kit from Molecular Devices (aka PMPI, plasma membrane potential indicator). The model used to calculate ΔψP assumes that the plasma membrane permeability for ions other than K+ is significant. The extracellular concentration of these ions is either constant, or are replaced for K+ (e.g. Na+). Furthermore, the model is also compatible by the presence of a constant voltage offset injected by electrogenic ion pumping during K-steps.
A limitation of this approach is the larger error committed during the fit than using the linear, Nernstian approach.
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 3 additions or replacements with high K+-medium (or 2 with no error estimation) and finally complete depolarization.
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.