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Sapphire Laser (Coherent) tuned at 920 nm. Emitted fluorescence was split with dichroic mirrors (Semrock) and filtered with appropriate filters (Semrock) for each channel before collection with nondescanned detectors. Typically, images from 15 to 20 z planes spaced by 5 µm were collected every 2 minutes for up to 3 hours. For in vitro analysis of PercevalHR-expressing BMDMs, two-photon imaging was performed using a 25X/1.05 NA objective (Olympus) installed into a FVMPE-RS upright microscope (Olympus) equipped with an Insight deep see dual laser (Spectra physics) and a resonant scanner. PercevalHR excitation was achieved using llow = 830 nm and lhigh = 1040 nm. Emitted fluorescence, collected sequentially for each l, was split with dichroic mirrors (Semrock) and filtered with a 520/35 filter (PercevalHR signal) and a 483/32 filter (background) before collection with GaAsP detectors. Images in a single plan were collected every 15 s for 5 to 10 min. Data collected were analyzed and processed using Fiji (ImageJ) and Imaris software. Quantification and statistical analysis Data are reported as the mean ± SD, and numbers of experiments are reported in figure legends. For in vitro analyses, statistical differences between two groups were evaluated using a two-tailed unpaired Student's t-test with Welch's correction or using an ordinary one-way ANOVA with post hoc Holm-Sidak test for multiple comparison. For in vivo analyses, unless indicated otherwise, statistical differences between two groups were evaluated using a Mann-Whitney U test. Correlation between the density of iNOS competent cell and cellular respiration and cytokine production was further analyzed in vitro and in vivo by exponential one-phase decay regression, Nature, vol.375, pp.408-411, 1995. ,
, Key Resources Table REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Purified anti-mouse CD16/32 (Fc-block) (clone: 93) BioLegend Cat#101302; RRID: AB_312801 Biotin anti-mouse TER-119/Erythroid cells (clone: TER-119) BioLegend Cat#116203, vol.RRID, p.313705
, RRID: AB_2651134 BV421 anti-mouse MHC II (I-A/I-E) (clone: M5/114.15.2) BioLegend Cat#107632; RRID: AB_2650896 BV510 anti-mouse Ly-6G/Ly-6C (Gr-1) (clone: RB6-8C5) BioLegend Cat#108438; RRID: AB_2562215 BV510 anti-mouse Ly-6G (clone: 1A8) BioLegend Cat#127633, RRID: AB_2562937 BV605 anti-mouse Ly-6C, pp.30-41, 128036.
, Alexa Fluor 488 anti-mouse TNF-a (clone: MP6-XT12) BioLegend Cat#506313, vol.RRID, p.493328
, Alexa Fluor 488 anti-mouse Ly-6G (clone: 1A8) BioLegend Cat#127626; RRID: AB_2561340 PE anti-mouse/rat/human MCP-1 (clone: 2H5) BioLegend Cat#505904, vol.RRID, p.315410
5 anti-mouse/human CD11b (clone: M1/70) BioLegend Cat#101228; RRID: AB_893232 PE anti-mouse CCL3 (MIP-1alpha) (clone: DNT3CC) Invitrogen Cat#12-7532-80, vol.RRID, p.2572661 ,
, 1 (clone: A20) BioLegend Cat#110730; RRID: AB_1134168 eFluor 660 anti-mouse CCL3 (MIP-1 alpha) (clone: DNT3CC) eBioscience Cat#50-7532-82, PE/Cy7 anti-mouse CD45, vol.RRID, p.2574295
, BioLegend Cat#117310; RRID: AB_313779 APC anti-mouse IL-1b Pro-form (clone: NJTEN3) eBioscience Cat#17-7114-80, APC anti-mouse CD11c (clone: N418), vol.RRID, p.1272175
, APC-eFluor 780 anti-mouse MHC II (I-A/I-E) (clone: M5/114.15.2) eBioscience Cat#47-5321-82, vol.RRID, p.1548783
, Alexa Fluor 488 Rat IgG1, kappa isotype ctrl (clone: RTK2071) BioLegend Cat#400417, vol.RRID, p.389319
IgG isotype ctrl (clone: HTK888) Biolegend Cat#400908; RRID: AB_326593 PE Rat IgG2a, kappa isotype ctrl (clone: RTK2758) Biolegend Cat#400508, vol.RRID, p.326529 ,
APC Rat IgG1 isotype ctrl (clone: eBRG1) eBioscience Cat#17-4310-82; RRID: AB_470178 Biological Samples Leishmania major, strain: LRC-L137 V121, DsRedexpressing parasites, 2000. ,
, , 2003.
, Chemicals, peptides, or recombinant proteins Lipopolysaccharide from Escherichia coli O26:B6 Sigma-Aldrich Cat#L2654
, Mouse IFN gamma Recombinant Protein Carrier-Free eBisocience Cat#34-8311-85
, , pp.98059-61
Cayman Chemical Cat#80310, CAS, vol.ID, pp.159190-159235 ,
Chemical Cat#11342; CAS ID: 579-13-5 SNAP Cayman Chemical Cat#82250, CAS, vol.ID, pp.67776-67782 ,
, Oligomycin A Cayman Chemical Cat#11342, CAS, vol.ID, pp.579-592
, MitoTracker Green FM Molecular Probes Cat#M7514, CAS, vol.ID, pp.201860-201877
, Sodium azide 10% solution Interchim Uptima Cat#NJK63A, CAS, vol.ID, pp.26628-26650
, MitoTracker Red CMXRos Molecular Probes Cat#M7512 Liberase TL Research Grade 10 mg Sigma, p.05401020001
Protein Transport Inhibitor) BD Biosciences Cat#555029 2-NBDG Sigma-Aldrich Cat#72987, CAS, vol.ID, pp.186689-186696 ,
, PBS Gibco Cat#13151014 Perm/Wash Buffer BD Biosciences Cat#554723 RIPA buffer Sigma-Aldrich Cat#R0278 cOmplete TM Protease Inhibitor Cocktail Sigma-Aldrich Cat#11697498001 XF Base Medium Minimal DMEM Seahorse Bioscience
, Recombinant Murine SCF Peprotech Cat#250-03
, Recombinant Murine Flt3-ligand Peprotech Cat#250-31L
, Recombinant Murine IL-3 Peprotech Cat#213-13
, Polybrene Transfection Reagent Merck Millipore Cat#TR-1003-G Freund's Adjuvant, Incomplete Sigma-Aldrich Blue Dead Cell Stain Kit Invitrogen Cat#L23105 jetPRIME Polyplus-transfection Cat#114, p.7
, Zombie Violet FM Fixable Viability Kit BioLegend Cat#423114 CD11c MicroBeads UltraPure, mouse Miltenyi Biotec, pp.130-108
, Seahorse XF96e FluxPak mini Seahorse Bioscience
, Seahorse XF Cell Mito Stress Test Kit Seahorse Bioscience Cat#103015-100
, Stress Test Kit Seahorse Bioscience
, Experimental Models: Cell Lines HEK 293 cells N/A N/A Experimental Models: Cell strains Mouse: C57BL/6J Charles River France JAX:000664 Mouse: C57BL/6J-Ptprc, 1975.
, 2654931 Mouse: C57BL/6-Tg(Csf1r-EGFP
, , p.3051865, 2004.
, Mouse: B6.129P2-Nos2 tm1Lau, p.1857228, 1995.
Addgene plasmid #49082 pCL, Image J) Schindelin et al, vol.10, 2012. ,
, GraphPad Software
, WT or Nos2 -/-BMDMs were activated 24 h with LPS+IFN-? either alone (ratios 100:0 and 0:100) or mixed at different ratios (50:50 and 10:90). (D-E) WT (CD45.1) or Nos2 -/-(CD45.2) BMDMs were activated 24 h with LPS+IFN-? either alone (ratios 100:0 and 0:100) or mixed at different ratios (50:50 and 10:90) and loaded with MitoTracker GreenFM and MitoTracker CMXRos. (D) Mitochondrial activity was normalized to the value of activated WT (100:0 ratio) for each group and graphed as a function of the density of iNOS competent cells in the culture. (E) Bar plots showing the normalized mitochondrial activity for the different mixed culture conditions. The inset shows the analysis of WT (CD45.1) and Nos2 -/-(CD45.2) cells in mixed cultures at the indicated ratio. (F-G) Percentages of cytokine-producing cells were assessed by intracellular cytokine staining for pro-IL-1b and CCL2. (F) Percentages of cytokine producing cells were graphed as a function of the density of iNOS competent cells in the culture. (G) Bar plots showing the percentages of producing cells for pro-IL-1b (top panel) and CCL2 (bottom panel) for the different mixed culture conditions, The ratio between MitoTracker CMXRos gMFI and MitoTracker GreenFM gMFI was calculated for each condition. Results are shown as fold change for the activated compared to the unactivated condition for (A) WT and Nos2 -/-cells or (B) untreated or L-NIL-treated WT cells. (C)
, 3D volume reconstruction was used to determine GFP + cell numbers and the corresponding cell densities at the site of infection. (D) CD45.1 WT recipient mice were lethally irradiated and reconstituted with CD45.1 WT and CD45.2 Nos2 -/-bone marrow cells, mixed at different ratios to modulate the tissue density of iNOS competent cells. Chimeras were infected 6 weeks later with DsRed-expressing L. major. Monocyte-derived cells activity was assessed 17 days later by intracellular cytokine staining on extracted ear cells. Percentages (top) and gMFI (bottom) of pro-IL-1b-, TNF-a-and CCL3-producing Ly6C + MHC-II + monocyte-derived cells, GFP + cells used to reconstitute chimera recipients. (C)