Differentiated J2 HIE monolayers (incubated with virus/VLPs for 10 min; bafilomycin for 1 h) were incubated with 50 nM LysoTracker (ThermoFisher Scientific) for 10 min. The cells were washed twice with CMGF(-) and LysoTracker staining of acidic compartments in treated and untreated J2 HIEs (mock) was observed by epifluorescence microscopy using Olympus cellSens Standard Version 2.3 software. Endocytosis measurements were carried out using FM1-43FX (ThermoFisher Scientific). HIE monolayers were treated media containing 10 μg/mL of FM1-43FX with or without VLPs for 10 min at 37 °C. Endocytosis was stopped by washing with prechilled PBS and the cells were fixed in 4% PFA for 20 min and nuclei were stained with 300 nM DAPI for 5 min at room temperature. Quantitation of the fluorescence was done using J-image. Briefly, every experiment was repeated at least three times with 3–4 images analyzed per condition in each experiment. Identical elliptical regions-of-interests (ROIs) were drawn per field and mean fluorescence intensity from these ROIs was measured.
Lysotracker
Manufactured by Olympus
LysoTracker is a fluorescent dye used to label and visualize acidic organelles, such as lysosomes, in living cells. It enables the tracking and analysis of these cellular compartments.
Automatically generated - may contain errors
Lab products found in correlation
Lysotracker, by Thermo Fisher Scientific (2 mentions)
Cellsens standard version 2, by Olympus (1 mentions)
Fm 1 43fx, by Thermo Fisher Scientific (1 mentions)
Fv10 asw 4.2 viewer software, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Fv1200 confocal microscope, by Olympus (1 mentions)
2 protocols using lysotracker
1
Visualizing Endocytosis in J2 HIE Monolayers
2
Macrophage Phagocytosis of E. coli
Check if the same lab product or an alternative is used in the 5 most similar protocols
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To evaluate whether TTP mediates the phagocytotic efficiency of macrophages, RAW264.7 cells (1 3 10 5 per well) were seeded in confocal 4 well Lab-Tek chambered coverglass (Nunc, Thermo Scientific, Waltham, MA). Cells were first transfected with scramble RNA (scRNA) or siRNA against TTP (siTTP) for 36 h. After that, cells were treated with CORM2 (20 mM) for 6 h. Alexa Fluor 488-labeled bioparticle E. coli (2 3 10 6 per well, Invitrogen) and 0.2 mM lysotracker (Life Technology) were added in cells and incubated in the dark at 37 C for 50 min. Then cells were fixed with 4% paraformaldehyde in PBS at room temperature for 30 min, and stained with 1 mg/ml DAPI (Sigma) for 15 min. Samples were washed with 1 3 PBS (GIBCO, Grand Island, NY) for 3 times, and were imaged with an Olympus FV1200 confocal microscope (Olympus, Tokyo, Japan). To analyze the phagocytosis of RAW 264.7 macrophages, we applied FV10-ASW 4.2 viewer software (Olympus, Version 4.2a) to observe the co-localization of E. coli (green) and lysotracker (red) and utilized Pearson's Correlation Coefficient for data analysis (n = 16 cells per group).
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