I am working with two fish cells types, a zebrafish liver cell line and primary cultures of trout macrophages. We are using lysotracker to stain them in order to visualize lysosomes in confocal microscopy, which is working with the liver cells but not with macrophages.
Has anyone had staining problems with lysotracker before, with live cells? Did it also depend on the cell type/line?
If so, what alternative did you use to stain the lysosomes?
Thank you for your time! =)
Dear Irene!
Please You look at following article:
https://www.nature.com/articles/s41598-020-57501-0
Zebrafsh C-reactive protein isoforms inhibit SVCV replication by blocking autophagy through interactions with cell membrane cholesterol
The adult XL wild type zebrafish of 700–900 mg body weight (3–4 cm long)
Evaluation of intracellular pH changes
In order to evaluate if CRPs can change the pH of the lysosomes in the ZF4 cell line, cells were incubated with the cell permeable green dye LysoTracker Green DND-26 (Cell Signaling Technology, Danvers, MA, USA), which stains acidic compartments (lysosomes) in live cells. Briefly, ZF4 cells were co-transfected with each crp-encoding plasmid (CRP-mix) or with just the empty plasmid (pMCV1.4) using the same methodology described above but seeding the cells into 24-well plates. After 48 h post-transfection, monolayers were incubated with 60 nM Lysotracker Green DND-26 for 30 min at 28 °C in dark conditions. Thereafter, monolayers were washed with PBS and then trypsin-detached. The fluorescence of each condition was measured by flow cytometry (FACSCalibur flow cytometer, BD Biosciences, San Jose, CA, USA). Each treatment was evaluated in 6 different samples.
Dear Irene!
Please You look at following article:
https://www.nature.com/articles/s41598-020-57501-0
Zebrafsh C-reactive protein isoforms inhibit SVCV replication by blocking autophagy through interactions with cell membrane cholesterol
The adult XL wild type zebrafish of 700–900 mg body weight (3–4 cm long)
Evaluation of intracellular pH changes
In order to evaluate if CRPs can change the pH of the lysosomes in the ZF4 cell line, cells were incubated with the cell permeable green dye LysoTracker Green DND-26 (Cell Signaling Technology, Danvers, MA, USA), which stains acidic compartments (lysosomes) in live cells. Briefly, ZF4 cells were co-transfected with each crp-encoding plasmid (CRP-mix) or with just the empty plasmid (pMCV1.4) using the same methodology described above but seeding the cells into 24-well plates. After 48 h post-transfection, monolayers were incubated with 60 nM Lysotracker Green DND-26 for 30 min at 28 °C in dark conditions. Thereafter, monolayers were washed with PBS and then trypsin-detached. The fluorescence of each condition was measured by flow cytometry (FACSCalibur flow cytometer, BD Biosciences, San Jose, CA, USA). Each treatment was evaluated in 6 different samples.
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