Mitochondrial membrane potential assay - measurement of spectral crossbleed

Parameters:

Name	#	Type	Description
Channel Number, TMRM	1	integer	Channel number associated with the image, as appears in the Multi-Dimensional Open Dialog. Use Edit/Rename to view or change channel number.
Channel Number, FLIPR	2	integer	Channel number associated with the image, as appears in the Multi-Dimensional Open Dialog. Use Edit/Rename to view or change channel number.
Background level (percentile)	3	real	Background is calculated as frame-by-frame median of those pixels that are the darker than this percentile of image histogram for the duration of the entire recording.
Direction of crossbleed (1st:TMRM, 2nd: FLIPR)	4	string	Which channel bleeds into the other in the order of selection as Image A. Available directions: "first->second","second->first". Internal to this pipeline, TMRM is always taken as first and FLIPR as second.

Description:

Recordings with tetramethylrhodamine methyl ester (TMRM) and FLIPR (aka PMPI, plasma membrane potential indicator; FLIPR Membrane Potential Probe) will be contaminated with fluorescence crossbleed, more or less, depending on the actual optical setup of the microscope. Mostly FLIPR fluorescence contaminates the TMRM signal. Spectral crossbleed can be calculated most precisely using single-fluorophore stained samples. This means two samples, one incubated with TMRM in basal condition, and an other incubated with FLIPR and then with CDC (or PM-PFA; see the ΔψM assay). It is also possible to calculate it from an actual potentiometric recording because the two fluorophores behave differently in time, but this method is less robust and typically allows only calculation of the stronger FLIPR to TMRM crossbleed. This pipeline is supported by an interactive protocol in the Primer.

The input is a two channel recording acquired with the same microscopy settings as the potentiometric recording, so a TMRM and a FLIPR channel. Single or multiple frames. The potentiometric medium is prepared either only with TMRM or on with FLIPR added. For FLIPR also a PM-PFA is prepared and added to the sample ~30 min before recording.

• Adjust channel numbers and the percentile level of background subtraction pipeline parameters to match the settings as you analyze the potentiometric time lapse recordings.
o If the cells are very sparse Use background level of 50 percentile.
o If the culture is sub confluent Use background level of 20 percentile.
o If the culture is confluent Use background level of 5 percentile.
• Select the direction of crossbleed:
o For TMRM-loaded sample: first->second
o For FLIPR-loaded sample: second->first
• Run the pipeline, and observe the resultant Excel worksheet.
o The crossbleed coefficient for "first->second" channel appears in the bottom left corner of the matrix.
o The crossbleed coefficient for "second->first" channel appears in the top right corner of the matrix.
• Run the pipeline in all relevant positions (e.g. use the pull-down menu of the button to toggle mode for partial plate operation) and calculate the median of the coefficients in Excel independently for the two ways of crossbleed.
• Enter the spectral crossbleed matrix as ={{1,second->first},{first->second,1}} in the Mitochondrial membrane potential measurement (TMRM/FLIPR).
• Save the data by File/Save Excel Data if needed.