Lysotracker green

Cell death was detected using LysoTracker, Nile Blue, or TUNEL, as previously described [17 (link), 26 (link), 27 (link)]. LysoTracker labels the increased lysosomal activity detected in dying cells and around phagocytosed cell debris [28 (link), 29 (link)] and has been used to identify the cell death in limb buds of various tetrapods, including X. laevis and coqui frogs [17 (link), 30 ]. Briefly, tadpoles at stages 32–37 were incubated with 0.5 μM LysoTracker Red (Thermo Fisher Scientific, Waltham, MA) in PBS+ (PBS pH 7.4, 9 mM CaCl₂, 3.3 mM MgCl₂) for 2 hours, washed and photographed with na LSM780 confocal microscope (Zeiss). Some of the LysoTracker Red-stained tadpoles were then stained with 0.01% Nile blue in filtered water for 20 minutes, washed, and photographed. For TUNEL staining, hindlimb buds from stage 36 tadpoles were cryosectioned at 8–12 μm as described [17 (link)], and stained using TUNEL Mix (In situ Cell Death Kit, Roche) according to the manufacturer’s protocol. For detection of cell death and reactive oxygen species, tadpoles at stage 36–37 were incubated with 0.5 μM LysoTracker Green and with 2 μM CellROX Deep Red (Invitrogen) in PBS+ for 2 hours, washed, and photographed with an LSM780 confocal microscope (Zeiss).

We used confocal microscopy to characterize the subcellular localization of MB. To this end, we compared the fluorescence arising from cell cultures simultaneously incubated in the presence of MB and standard fluorescent markers of organelles. MitoTracker Green (Invitrogen, Paisley, UK) was used as a mitochondrial marker, LysoTracker Green (Invitrogen) as a lysosome marker and HO as a marker for the cell nucleus. Confocal images were taking using a laser scan microscope (LSM) - 510 from Zeiss using 1.2 N.A. 40x water immersion or 1.4 N.A. 63x oil immersion objective lenses. The laser and filter settings were: laser lines for MB = 633, Lyso = 488 and Hoechst 33342 = 364; beam splitter = HFT UV/488/543/633; emission filters for MB: 651-704, Lyso = 501-554 and Hoechst 33342 = 435-485. The imaging settings were: zoom = 1, dimensions = 512x512 pixels, image depth = 16 bit, averaging signal = 2 and optical section thickness = 2 μm. Images had their brightness and contrast adjusted for the figures and were analysed with ImageJ Software (National Institutes of Health).

The neutral rhenium(I) molecular probe ReZolve-L1^™ was prepared according to a previously published procedure [41 (link)]. ReZolve-L1^™ was dissolved in DMSO to prepare a 10 mM stock solution, which was diluted in PBS to 10 μM for tissue staining. For Raman confocal microscopy, 3T3 cells were fixed using cold methanol and air dried [37 (link)] before being incubated with ReZolve-L1^™ overnight. Fat body tissue was dissected from Drosophila larvae or pupae into PBS. For Raman confocal microscopy, the tissue was fixed in 4% paraformaldehyde (Sigma Aldrich, St Louise, USA) ready for mapping. For live confocal and two photon microscopy, the fat body tissue was incubated with ReZolve-L1^™ for 15 minutes at room temperature and washed in PBS for 30 seconds. Tissues were counterstained for acidic compartments with LysoTracker^® Green (prepared according to manufacturer instructions; Invitrogen, USA) for 2 minutes at room temperature. Tissues were then mounted in carbomer-940 (Snowdrift farm, Tucson, USA) based optical coupling gel to prevent dehydration prior to imaging; see protocol in [97 (link)]. For Oil Red O staining, fat body tissue was fixed in 2% (v/v) paraformaldehyde for 20 minutes at room temperature. Tissues were then washed three times in PBS for ten minutes before incubation in 1/100 Oil Red O for 30 minutes and then washed in PBS before mounting in 80% glycerol.

Confocal microscopy sample were analyzed with an Olympus FV10i Spectral Confocal microscope. Two million MDMs were cultured on 12 mm glass cover slips in 24-well tissue culture plates and infected synchronously with k56-2 at an MOI of 2 or 10. Nuclei were stained with the nucleic acid dye 4′,6′-diamino-2-phenylindole (DAPI) blue for imaging. LC3 stained green with a cleaved LC3 antibody detection (Abgent, AP1805a). Lysosomes were stained green with Lysotracker Green (Invitrogen, L7526). p62 was detected with a green fluorescent ligand (BD Bioscience, 610832). At least one hundred macrophages were scored for each condition with scoring verified by independent study members. All experiments were performed in at least triplicate.

Cultured cells were treated with AlbiCpG for 2 h and washed with PBS. Endolysosome was stained with LysoTracker Green (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction. Cells were immersed in HEPES buffer (pH 7.4) during imaging. Super-resolution imaging was conducted on an instant linear structured illumination microscope (instant SIM) (built in Dr Hari Shroff Laboratory)^{41 (link)}. On iSIM, Alexa488-labeled AlbiCpG (Ex: 488 nm) and LysoTracker Red DND-99 (Ex: 561 nm) was used for imaging for a long duration. Images were deconvolved and analyzed using home-built software^{41 (link)}. Alternatively, confocal microscopy with deconvolution was conducted on a Leica SP8 workstation (Leica Microsystem, IL). On the Leica SP8 workstation, MEB fluorescence was monitored to image AlbiCpG (Ex: 561 nm; Em: 600–670 nm), confirming that there was minimal overlap in fluorescence between LysoTracker Green and MEB. Raw imaging results were deconvolved and analyzed on the Leica SP8 workstation.