Primary ATII cells were incubated with either 10 µM BODIPY FL ATP or 10 µM mant-ATP (Thermo Fisher Scientific) for 3 h in MucilAir medium, after 2.5 h LTR (100 nM) was added to the medium. Cells were then washed three times with bath solution (and maintained in bath solution) and immediately mounted on a Cell Observer inverse microscope (Zeiss) for analysis of fluorescence. All images were acquired at identical settings (e.g., magnification, gain, excitation). Mean fluorescence of individual LBs was analyzed within regions encircling individual LBs (see Fig. 4 A and Fig. S5).
Bodipy fl atp
Manufactured by Thermo Fisher Scientific
Sourced in United States
BODIPY FL ATP is a fluorescent analog of adenosine triphosphate (ATP). It is a useful tool for studying ATP-dependent processes in biochemical and cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Mant atp, by Thermo Fisher Scientific (1 mentions)
Cell observer, by Zeiss (1 mentions)
Lysotracker red, by Thermo Fisher Scientific (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (1 mentions)
Thioltracker violet, by Thermo Fisher Scientific (1 mentions)
Synergy neo2, by Agilent Technologies (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Slot blot apparatus, by Hoefer (1 mentions)
6 protocols using bodipy fl atp
1
Imaging Lipid Droplet Fluorescence
2
Chemical Reagents for Cell Assays
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All chemicals and reagents used in this study were purchased from Sigma-Aldrich Chemical Company (Shanghai, China), except for those specifically mentioned. Tissue culture medium-199 (TCM-199), Dulbecco's phosphate buffered saline (DPBS), Dulbecco's modified Eagle's medium (DMEM), knockout serum replacement, CM-H2DCFDA, ThiolTracker™ Violet, MitoTracker™ Red CMXRos, BODIPY FL ATP, LysoTracker™ Red were obtained from ThermoFisher Scientific (Shanghai, China).
3
ATP Labeling of 4-cell Embryos
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Denuded 4-cell embryos were washed three times in PBS-PVA, fixed with 4% paraformaldehyde-PBS for 1 h, washed three times, and then incubated in PBS-PVA supplemented with 500 nmol/L BODIPY FL ATP (A12410; Molecular Probes, Eugene, OR, United States) for 1 h at room temperature in the dark (40 (link)). Four-cell embryos were washed three times in PBS-PVA and captured using an epifluorescence microscope (EVOS™ FL Auto; Life).
4
ATP Staining of 4-Cell Embryos
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Denuded 4-cell embryos were washed three times in PBS-PVP and xed with 4% paraformaldehyde-PBS for 1 hour, washed three times, and incubated in PBS supplemented with 500 nM BODIPY FL ATP (A12410; Molecular Probes, Eugene, OR, USA) for 1 hour at room temperature in the dark. 4-cell embryos were washed three times in PBS and were captured using uorescence microscopy imaging system.
5
Rad50 ATP Binding Affinity Assay
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Pf MR complex ATP binding affinity was measured as previously described (23 (link)) with slight modification. Briefly, 5 nM Bodipy FL ATP was titrated with either 0–25 μM MRNBD (i.e. 0–50 μM Rad50 ATP binding sites) or 0–22.5 μM full-length MR. Fluorescence polarization of Bodipy FL ATP (Life Technologies) was measured at room temperature, to limit ATP hydrolysis, in a Synergy Neo2 multi-mode reader (BioTek) using a FP 485/530 filter. Binding affinities were calculated by fitting polarization (F) versus protein concentration ([P]) to the two-state quadratic binding function, where F0 and Fmax are the initial and final polarization values, [ATP] is the concentration of fluorescent ligand, and KD is the calculated dissociation constant. Errors are the standard deviation of at least three experiments.
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Pf MR complex ATP binding affinity was measured as previously described (23 (link)) with slight modification. Briefly, 5 nM Bodipy FL ATP was titrated with either 0–25 μM MRNBD (i.e. 0–50 μM Rad50 ATP binding sites) or 0–22.5 μM full-length MR. Fluorescence polarization of Bodipy FL ATP (Life Technologies) was measured at room temperature, to limit ATP hydrolysis, in a Synergy Neo2 multi-mode reader (BioTek) using a FP 485/530 filter. Binding affinities were calculated by fitting polarization (F) versus protein concentration ([P]) to the two-state quadratic binding function, where F0 and Fmax are the initial and final polarization values, [ATP] is the concentration of fluorescent ligand, and KD is the calculated dissociation constant. Errors are the standard deviation of at least three experiments.
6
Quantifying FtsA ATP Binding Dynamics
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