Mitotracker red

MitoTracker staining was performed as described in Gaffney et al. (2014) (link). Briefly, 20 nematodes were incubated with 20 μl of 4.7 μM MitoTracker Red (Thermo Fisher Scientific, Waltham, United States). Following 1 hr incubation, nematode suspension was washed in M9 buffer for three times. Animals were then imaged using a confocal microsope (Zeiss LSM800) at 20X magnification, at a wavelength of 540/25 nm.

GFP-LC3-HeLa cells were collected and inoculated in 6-well plates at a density of 0.5 × 10⁵ cells/mL. The cells were incubated for 24 h followed by the addition of ATO and Met and incubation for a further 6 h. Mito tracker red (Thermo Fisher Scientific, Massachusetts, USA), 1 mmol/L was then added to each well to a final concentration of 500 nmol/L and incubated at 37°C for 30 min. The distributions of red and green fluorescence in the cells were observed by laser confocal microscopy.

Mitotracker Red (ThermoFisher #M22425) was resuspended in DMSO (0.25 mM) and diluted to 200 nM in E3 embryo medium. nco and csr embryos were then incubated in the dark for 20 minutes before removing Mitotracker solution and replacing with fresh E3 embryo medium. Samples were allowed to stabilize in the dark for 30 minutes before imaging at 21 hpf. Embryos were then phenotyped at 27 hpf.

Hoechst 33258, and calcium ionophore III were obtained from Sigma (St Louis, MO, USA). Acridine orange (AO)/ethidium bromide (EB) double stain assay kit, L-glutathione (Reduced) and tunicamycin were purchased from Solarbio Science & Technology Co. (Beijing, China). Protein A/G-agarose was from Biogot Technology Co. (Nanjing, Jiangsu, China). Lipofectamine RNAiMAX and Lipofectamine 2000 were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Pronase was from Roche Diagnostics GmbH (Mannheim, Germany) and recombinant histone H3 was purchased from Active Motif (CA, USA). LysoTracker, MitoSox, MitoTracker Green, and MitoTracker Red were purchased from Thermo Fisher Scientific (Waltham, Ma, USA). ER-Red and DIOC6 (3) were acquired from Nanjing KeyGen Biotech Co. (Nanjing, Jiangsu, China). Fluo 3-AM special packaging was purchased from Dojindo Laboratorie (Kumamoto-ken, Kyushu, Japan). Thapsigargin was obtained from ACROS Organics (Belgium). LysoSensor^TM Yellow/Blue DND-160 (PDMPO) was purchased from Yeasen (Shanghai, China). DO₂, NaOD, and DCl were acquired from J&K Scientific (Beijing, China). R18 peptide was obtained from Enzo Life Sciences (New York, USA).

Propidium iodide (PI) staining was used to detect nucleic acid and cell viability. Following fixation (as described above), nucleic acid staining was performed by incubating the samples in PI (1 μM) in PBS for 30 minutes. Samples were counter stained with DAPI and a no point were treated with RNase. Alternatively, for cell viability, cells were incubated with the PI solution prior to fixation.
The membrane marker wheat germ agglutinin (WGA) conjugated with Alexa Flour 488 (Life technologies) was used as an additional cell marker. Post-fixation, cells were incubated in a 10μg per mL solution for 30 minutes.
Protein synthesis was measured with Click-it HPG Alexa Fluor 488 protein synthesis assay (Thermo Fisher, C10428) according to manufacturer’s instructions. As a negative control, cells were treated with cycloheximide (50μg/mL) during the protein synthesis incubation.
Fluorescent detection of mitochondria was conducted with MitoTracker Red (Thermo Fisher, M7512). Staining was performed on live cells and fixed for imaging, per the manufacturer’s instructions.
Phagocytosis assay used green fluorescent beads 1 micron in size, and were a generous gift from the Berwin Lab (Dartmouth College). Hemolymph from 5 larvae was incubated with 1 μl of beads for 1 hour prior to imaging.