To assess the levels of G-actin in the cytoplasm, the DNase I/phalloidin staining method was used17 (link), 18 (link). Briefly, cells treated with 25 µM BBS at different time points were fixed in 4% formaldehyde (EM grade) with 0.32 M sucrose in the cytoskeleton buffer (10 mM MES, 138 mM KCl, 3 mM MgCl2, 4 mM EDTA, pH 6.1) for 10 min at 37°C. After permeabilization followed by blocking with 3% BSA/PBS, the samples were incubated with 0.3 µM Alexa Fluor® 488-DNaseI and 1:2000 Alexa Fluor® 568-conjugated phalloidin (Molecular Probes). Confocal z-stacks upon subtraction of the background fluorescence were used to measure total integrated fluorescence of DNaseI and phalloidin in individual cells. To avoid changes in G-actin levels associated with cell size heterogeneity, DNaseI and phalloidin fluorescence levels were normalized per cell size. In our experiments, an increase in G-actin levels (DNaseI fluorescence) was always associated with a decrease in F-actin levels (phalloidin fluorescence).
Alexa fluor 488 dnasei
Manufactured by Thermo Fisher Scientific
Alexa Fluor® 488-DNaseI is a conjugate of the Alexa Fluor® 488 dye and the DNase I enzyme. DNase I is an endonuclease that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone. The Alexa Fluor® 488 dye provides a fluorescent label for the DNase I enzyme.
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Lab products found in correlation
3 protocols using alexa fluor 488 dnasei
1
Quantifying Cytoplasmic G-Actin Levels
2
Visualizing Legionella Infection Dynamics
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F- and G-actin were visualized from Legionella-infected macrophages using Alexa Fluor® 568 phalloidin and Alexa Fluor® 488 DNAse I (Molecular Probes) and for studies examining colocalization of GFP-expressing bacteria (Legionella and E. coli) with the lysosome, Lysotracker™ red was used to stain acidic vesicles of infected BMDMs, as previously described14 (link). To examine cell death, after infection macrophages were stained with 4 uM ethidium homodimer-1 (EthD-1) from the LIVE/DEAD® viability/cytotoxicity kit (Molecular Probes), according to manufacturer’s instructions. Images were captured using laser scanning confocal fluorescence microscope with a 60X objective (Olympus Fluoview FV10i).
3
Quantifying Cytoplasmic G-Actin Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To assess the levels of G-actin in the cytoplasm, the DNase I/phalloidin staining method was used17 (link), 18 (link). Briefly, cells treated with 25 µM BBS at different time points were fixed in 4% formaldehyde (EM grade) with 0.32 M sucrose in the cytoskeleton buffer (10 mM MES, 138 mM KCl, 3 mM MgCl2, 4 mM EDTA, pH 6.1) for 10 min at 37°C. After permeabilization followed by blocking with 3% BSA/PBS, the samples were incubated with 0.3 µM Alexa Fluor® 488-DNaseI and 1:2000 Alexa Fluor® 568-conjugated phalloidin (Molecular Probes). Confocal z-stacks upon subtraction of the background fluorescence were used to measure total integrated fluorescence of DNaseI and phalloidin in individual cells. To avoid changes in G-actin levels associated with cell size heterogeneity, DNaseI and phalloidin fluorescence levels were normalized per cell size. In our experiments, an increase in G-actin levels (DNaseI fluorescence) was always associated with a decrease in F-actin levels (phalloidin fluorescence).
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