In vivo fluorescence lifetime imaging of macrophage intracellular metabolism during wound responses in zebrafish

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Tables

Table 1

Endpoints	Definition	Interpretation
Optical redox ratio Chance et al., 1979	${\displaystyle \frac{{\mathit{I}}_{NAD(P)H}}{{\mathit{I}}_{NAD(P)H}{\mathbf{+}\mathit{I}}_{FAD}}}$	Increase in optical redox ratio (ORR) = more reduced intracellular environment; likely increase in glycolysis; decrease in ORR = more oxidized intracellular environment; likely decrease in glycolysis. (I, intensity)
Nicotinamide adenine dinucleotide (phosphate) (NAD(P)H)	Short lifetime of NAD(P)H	Free/unbound NAD(P)H
NAD(P)H ${\displaystyle {\tau }_2}$	Long lifetime of NAD(P)H	NAD(P)H bound to a protein
NAD(P)H ${\displaystyle {\alpha }_1}$	Fractional component of free NAD(P)H	${\displaystyle {\alpha }_2}$ is fractional component of bound NAD(P)H, ${\displaystyle {\alpha }_1+{\alpha }_2=1}$; quantifies the pools of NAD(P)H in free and bound states
NAD(P)H mean lifetime ()	${\displaystyle {\tau }_m={\tau }_1{\alpha }_1+{\tau }_2{\alpha }_2}$	Weighted average of individual lifetime endpoints (); one can look at changes in individual endpoints to see what drives changes in ${\displaystyle {\tau }_m}$; for instance, a decrease in ${\displaystyle {\tau }_m}$ can be due to increase in ${\displaystyle {\alpha }_1}$, decrease in ${\displaystyle {\tau }_1}$, and/or decrease in ${\displaystyle {\tau }_2}$
Flavin adenine dinucleotide (FAD) ${\displaystyle {\tau }_1}$	Short lifetime of FAD	FAD bound to a protein
FAD ${\displaystyle {\tau }_2}$	Long lifetime of FAD	Free/unbound FAD
FAD ${\displaystyle {\alpha }_1}$	Fractional component of bound FAD	${\displaystyle {\alpha }_2}$ is fractional component of free FAD, ${\displaystyle {\alpha }_1+{\alpha }_2=1}$; quantifies the pools of FAD in free and bound states
FAD ${\displaystyle {\tau }_m}$	${\displaystyle {\tau }_m={\tau }_1{\alpha }_1+{\tau }_2{\alpha }_2}$	Weighted average of individual lifetime endpoints (${\displaystyle {\tau }_1,{\tau }_2,{\alpha }_1,{\alpha }_2}$); one can look at changes in individual endpoints to see what drives changes in ${\displaystyle {\tau }_m}$; for instance, a decrease in ${\displaystyle {\tau }_m}$ can be due to increase in ${\displaystyle {\alpha }_1}$, decrease in ${\displaystyle {\tau }_1}$, and/or decrease in ${\displaystyle {\tau }_2}$
Optical Metabolic Imaging (OMI) index Walsh and Skala, 2015	${\displaystyle \frac{OR{R}_i}{<ORR>}+\frac{NAD(P)H{\tau }_{mi}}{<NAD(P)H{\tau }_m>}-\frac{FAD{\tau }_{mi}}{<FAD{\tau }_m>}}$	Composite measure of mean-centered optical redox ratio and mean lifetimes of NAD(P)H and FAD; increase in the OMI index corresponds to increased redox ratio, and increased NAD(P)H and FAD protein-binding activities

Table 2

Control versus	2-Deoxy-d-glucose	Lm-infected wound TNFα−	Lm-infected wound TNFα+	Lm-infected wound	Burn wound24 hr	Burn wound72 hr
Optical redox ratio	↓	-	-	↓	↓	nd
OMI index	-	-	-	↓	↓	nd
NAD(P)H ${\displaystyle {\tau }_m}$	↓	↓	↓	↓	↓	↓
NAD(P)H ${\displaystyle {\tau }_1}$	nd	↓	↓	↓	↓	nd
NAD(P)H ${\displaystyle {\tau }_2}$	↓	↓	↓	↓	nd	nd
NAD(P)H ${\displaystyle {\alpha }_1}$	↑	nd	nd	↑	↑	↑

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Strain, strain background (10,403 S)	Listeria monocytogenes (strain 10,403 S)	PMID:26468080		Was be obtained from JD Sauer Lab, University of Wisconsin - Madison
Strain, strain background (Danio rerio)	WT (AB)	ZIRC	ZL1	https://zebrafish.org/home/guide.php
Strain, strain background (D. rerio)	Tg(tnf:GFP) (AB)	PMID:25730872		Was obtained from Michel Bagnat Lab, Duke University
Strain, strain background (D. rerio)	Tg(mpeg1:histone2b-GFP) (AB)	PMID:31259685		Can be obtained from Anna Huttenlocher Lab, University of Wisconsin - Madison
Strain, strain background (D. rerio)	Tg(mpeg1:mCherry-CAAX) (albino)	PMID:26887656		Was obtained from Leonard Zon Lab, Boston Children’s Hospital, Dana Farber Cancer Institute
Strain, strain background (D. rerio)	stat6 mutant (AB)	PMID:33761328		Was obtained from David Tobin Lab, Duke University
Chemical compound, drug	Metformin	Enzo Life Sciences, cat no ALX-270–432 G005	https://www.enzolifesciences.com/ALX-270-432/metformin/	1 mM in E3, bathing, start treatment at 1 day post fertilization, refresh daily
Chemical compound, drug	2-Deoxy-d-glucose	Sigma, cat no D8375	https://www.sigmaaldrich.com/US/en/product/sigma/d8375	5 mM in E3, bathing, 1 hr pretreatment right before imaging
Software, algorithm	GraphPad Prism		RRID:SCR_002798	https://www.graphpad.com/scientific-software/prism/
Software, algorithm	SAS		RRID:SCR_008567	https://www.sas.com/en_us/home.html
Software, algorithm	Fiji, ImageJ	Schindelin et al., 2012	RRID:SCR_002285	https://fiji.sc/
Software, algorithm	Cell profiler		RRID:SCR_007358	https://cellprofiler.org/
Software, algorithm	Matplotlib		RRID:SCR_008624	https://matplotlib.org/
Software, algorithm	R project for statistical computing	R Core Team, 2019	RRID:SCR_001905	https://www.r-project.org/
Software, algorithm	MATLAB R2019b			https://www.mathworks.com/
Software, algorithm	SPCImage 7.4			https://www.becker-hickl.com/products/spcimage/
Other	Line-powered thermal cautery instrument	Stoelting	59,005	https://stoeltingco.com/Neuroscience/Thermal-Cautery-Instruments~9879
Other	Type E tip for cautery instrument	Stoelting	59,010	https://stoeltingco.com/Neuroscience/Thermal-Cautery-Instruments~9879

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