Alexa488 dextran

For co-localization experiments cells were co-incubated with 250 nM folded aptamers and 50 μg/ml of Alexa488-transferrin (Invitrogen^™ by Life Technologies; Carlsbad, MA, USA) or 200 μg/ml Alexa488-dextran (Thermo Fisher Scientific Inc. Waltham, MA, USA). Subsequent washings, fixations and quenching steps were performed as described above.
For identification of endosome-like structures a co-staining was performed using aptamers or antibodies and Alexa488-transferrin. Cells were co-incubated with primary antibodies or aptamers as described above with 50 μg/ml Alexa488-transferrin for 1 hour at 37°C and 5% CO₂ in complete DMEM. Washing, fixation, permeabilisation and incubation of secondary antibodies were performed as described above.

Cell transplantation was performed as previously described (Masai et al., 2003 (link)). Wild-type zygotes were injected with Alexa-488 dextran (Thermo Fisher Scientific, D22910) and used for donor embryos. slbp1 mutant embryos carrying Tg[mfap4:tdTomato-CAAX] were used as host embryos. Host embryos carrying donor retinal cells were selected by observing Alexa 488 fluorescence at 24 hpf. slbp1 mutant and wild-type sibling embryos were sorted based on the slbp1 mutant morphological phenotype at 48 hpf and used for live imaging. After confocal images were obtained, the number of ocular mfap4-positive microglial precursors associated with Alexa-488 dextran-labeled donor transplanted retinal columns was counted. The fraction of ocular mfap4-positive microglial precursors associated with donor transplanted retinal columns in total ocular microglial precursors was calculated. The trapping efficiency of ocular mfap4-positive microglial precursors per transplanted donor retinal column was calculated using the total number of donor transplanted retinal columns in the retina. Detailed information on each transplanted eye is shown in Figure 5—figure supplement 1A-B.

NHBE cells seeded onto chamber slides were loaded for 3 hours with 20 μM Alexa 488 Dextran, 10kDa (Life Technologies) at 37˚C in a humidified incubator, prior to treatment with media only, 20 μg/ml LL-37 (or TAMRA-labeled LL-37), PAO1 at 10:1 MOI, or PAO1 + LL-37 for a further 3 hours. Cells were then imaged live using a Leica SP5 confocal microscope. Acquired images were subsequently analysed for evidence of lysomal leakage, indicated by a diffusion of the green fluorescent signal throughout the cell cytoplasm, compared to bright punctate fluorescence in media-only control conditions. Quantitation of leakage was performed by expressing the number of cells displaying diffused green fluorescence as a percentage of the total number of cells in that field, across a minimum of 5 fields per experiment.

Osteoclast precursors were cultured on bovine dentine slices in alpha-MEM supplemented with M-CSF and RANKL to induce osteoclast differentiation. Differentiated osteoclasts were incubated with 200 μg/ml aldehyde fixable Alexa 488-dextran (Life Technologies, Grand Island, NY, USA) overnight at 37°C. Cultures were fixed in 4% PFA for 15 minutes, counterstained with DAPI (4,6-diamidino-2-phenylindole) to visualize nuclei, and mounted on glass cover slips for fluorescence microscopy. Digital images (obtained using fluorescence microscope FSX100, Olympus) were processed with Image J to obtain an interactive 3D surface plot, where the height of the surface plot reflects image brightness, corresponding to the amount of internalized dextran. Multinucleated cells were counted and each cell was assessed for dextran endocytosis.

Cells were prepared in 48-well plates as described above. Then, cells were loaded with Alexa 488 Dextran (20 μM), 10 kDa (Life Technologies) for 3 h prior to LPS and CATH-2 treatment for further 6 h. After incubation, the cells were observed using fluorescence microscopy (Olympus, Tokyo, Japan).

HeLa or 3T3 fibroblasts (ATCC, Manassas, VA, USA) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) either from Lonza BioWhittaker (Walkersville, MD, USA) or from Corning (Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS, Fisher Scientific, Waltham, MA, USA) and antibiotics. To prepare hypoosmotic media, DMEM was diluted with water, or in some cases, with 10% FBS. Hyperosmotic media were prepared by adding sucrose to DMEM.
Osmolarity was not measured directly but estimated based on the manufacturer’s data for DMEM (340 mosm/kg for either source) and assuming ideal osmotic behavior. When 10% FBS, rather than water, was used to prepare dilutions, the numbers for osmolarity were adjusted accordingly.
The following reagents were used: gramicidin, fluorescein sodium, Hoechst 33258, DCPIB, and DiOC₁₈(3) (3,3′-dioctadecyloxacarbocyanine perchlorate; DiO), all from Sigma-Aldrich (St. Louis, MO, USA); torin 1 and rapamycin from ApexBio (Houston, TX, USA); 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS), amiloride, and cytochalasin D from Cayman Chemical (Ann Arbor, MI, USA); Acid Blue 9 (TCI America, Portland, OR, USA); NucView 488 (Biotium, Fremont, CA, USA); dextran Alexa 488 (Invitrogen, Eugene, OR, USA); tetramethylrhodamine ethyl ester (TMRE; Biotium, Hayward, CA, USA), and calcein AM ester (AnaSpec, Fremont, CA, USA).

CHO WT and CHO ΔXylT cells were seeded on coverslips for pulse-chase experiments. In brief, CHO cells were first incubated with dextran-Alexa568 (10,000 MW, 0.4 mg/mL) (Invitrogen) for 4 h, washed and incubated in dextran-free CHO medium for 18 h. Cells were then pulsed with dextran-Alexa488 (10,000 MW, 0.4 mg/mL) for 10 min, washed and incubated in CHO medium for 30 min. CHO WT and CHO ΔXylT cells were fixed with 4% PFA, and stained with phalloidin-iFluor 647 Reagent (1:1000) (Abcam) and DAPI. Images of 20 random FOVs were acquired on Zeiss Apotome.2 microscope with 63 × oil immersion objective using AxioVision 4.9.1 software (Zeiss). Spatial resolution of images was 9.7674 pixels per micron, pixel size: 0.1024 × 0.1024 micron². Endo-lysosomal fusion was scored by quantifying co-localization between the two labeled dextrans using ImageJ version 1.52e and JACoP plugin for pixel intensity spatial correlation analysis (37 (link)). Pearson’s correlation coefficient and Manders split coefficients (M1 and M2, thresholds set manually for both channels) were calculated.