The luciferin/luciferase detection of ATP was performed with the microplate reader Infinite F200 Pro (TECAN, Lyon, France) with ATP bioluminescent somatic cell assay kit (FLASC, Sigma, Saint-Quentin Fallavier, France). Breast cancer cell lines MCF-7, MDA-MB-231 and MDA-MB-435S transfected with siRNA control (siC) or siRNA targeting IP3R3 (siR3) were seeded at 2000 cells/well in white 96-well Nunc dishes with clear bottoms 72 hours before ATP measurements. For extracellular ATP measurements, 100μl of supernatant were incubated with the luciferin/luciferase (FLAAM, Sigma) at a final concentration of 0.04%. For intracellular ATP measurements cells were incubated with 100 μl of ATP releasing reagent (FLSAR, Sigma), before incubation with the luciferin/luciferase (FLAAM, Sigma). To determine the amount of ATP released, a calibration curve was constructed using known concentrations of ATP in solution (1, 10, 100, 1000, 10 000, 100 000 pM). Control experiments were performed to eliminate any drug effect on luciferase activity.
Flsar
Manufactured by Merck Group
Sourced in Switzerland
The FLSAR is a laboratory equipment product offered by Merck Group. It is a fluorescence detection system designed for sensitive and accurate analysis of samples. The FLSAR utilizes laser excitation and specialized detectors to enable reliable quantification and characterization of fluorescent molecules or compounds within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Atp bioluminescent somatic cell assay kit flasc, by Merck Group (1 mentions)
Flaam, by Merck Group (1 mentions)
Infinite f200 pro, by Tecan (1 mentions)
Atp colorimetric assay kit, by Abcam (1 mentions)
Victortm x3 multilabel plate reader, by PerkinElmer (1 mentions)
Bioluminescent somatic cell assay kit flasc, by Merck Group (1 mentions)
Luciferase, by Promega (1 mentions)
Disodium salt hydrate, by Merck Group (1 mentions)
Trichloroacetic acid, by Merck Group (1 mentions)
5 protocols using flsar
1
ATP Bioluminescence Assay in Breast Cancer Cells
2
Quantifying Cytoprotective Effects of H2S
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HL-1 cells were seeded in 12-well plates at 10,000 cells per well in complete Claycomb medium. Cells were preconditioned with GYY4137 over 12 hours and ATP content was analyzed periodically. To evaluate the cardio-protective effect of H2S during nutrient deprivation, HL-1 cells were preconditioned overnight with GYY4137 and the culture medium was then replaced with PBS to promote starvation. ATP content was analyzed at various times. To measure ATP, cells were washed twice with PBS buffer and then lysed with the addition of somatic cell ATP releasing reagent (FLSAR, Sigma-Aldrich). The supernatant was collected and kept at −20°C until analysis. ATP content was measured with the bioluminescent ATP somatic cells assay kit (FLASC, Sigma-Aldrich). Luminescence of triplicate samples was read on a VICTORTM X3 Multilabel Plate Reader (PerkinElmer) with a test wavelength of 570 nm. Rat hearts were explanted and stored at −80°C for ATP quantification with the colorimetric ATP Assay Kit (Abcam) following the manufacturer’s instructions. Briefly, tissue samples were homogenized in ATP assay buffer, deproteinized with 1 M perchloric acid, and ATP content was determined by absorbance at 570 nm. ATP levels from experimental samples were compared to sham samples (explanted hearts without treatment).
3
Measuring Oocyte ATP Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ATP levels in ovulated oocytes were measured using a Bioluminescent Somatic Cell Assay Kit (FL-ASC; Sigma-Aldrich), which makes use of the luciferin–luciferase reaction. Briefly, oocytes (18 oocytes from natural ovulation and 39 oocytes from hyperstimulation) were placed into 100 μl of ice-cold somatic cell reagent (FL-SAR; Sigma-Aldrich) for 5 min. They were then incubated with 100 μl of diluted ice-cold assay mix [FL-AAM reagent, diluted 1:25 in ATP Assay Mix Dilution Buffer (FL-AAB reagent; Sigma-Aldrich)] for an additional 5 min. The solution was then transferred to an AutoLumat LB 953 Luminometer (EG&G Berthold) for the measurement of luminescence. A 10-point standard curve (0–10 pmol/tube) was constructed for every 20 oocytes, and ATP content was calculated using the formula from linear regression of the standard curve.
4
Quantification of Tissue ATP and ADP
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen liver tissue was powdered, homogenized in HEPES buffer (25 mmol/L HEPES, 10 mmol/L MgCl 2 , 0.02% NaAc), and incubated for 10 minutes with 150 mL 6% trichloroacetic acid (Sigma, Buchs, Switzerland) and 100 mL ATP releasing reagent (FLSAR; Sigma). The supernatant was analyzed in a luminometer (Labsystems Luminoscan 1.2-0; Labsystems, Helsinki, Finland) after addition of 50 mL ATP monitoring reagent (Luciferase; Promega, Du ¨bendorf, Switzerland). ATP concentrations were calculated from a calibration curve using pure ATP (disodium salt hydrate; Sigma) for each experiment.
ADP/ATP ratios were determined using a ratio assay kit (Abcam, Lucerne, Switzerland, #ab65313) via bioluminescent detection.
ADP/ATP ratios were determined using a ratio assay kit (Abcam, Lucerne, Switzerland, #ab65313) via bioluminescent detection.
5
ATP Levels in HEK293T Cells Exposed to IP-1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ATP levels were determined 15, 30, and 60 min after the exposure of HEK293T cells to 10 and 50 μM of IP-1. ATP determinations were performed using previously described methodology [33 (link)]. Briefly, the extracellular medium of HEK cells was collected, the cells were washed twice with pre-warmed Locke’s solution containing (in mM): 154 NaCl, 5.6 KCl, 3.6 NaHCO3, 2.3 CaCl2, 5 HEPES, 5.6 glucose, pH 7.4, and lysed by incubation in 60 μL somatic cell ATP releasing agent (Sigma FL-SAR). 10 μL of lysates or 100 μL of extracellular medium were used to measure ATP levels by means of a luminometer (Bio Orbit) using the luceferin-luciferase Chemiluminiscent kit (Molecular Probes A22066). The luminometer records quimioluminescence values in millivolts and ATP concentrations were calculated from readings obtained from an ATP standard curve (1.95–250 pmol). Aliquots of cell homogenates were kept for protein determination by the micro-Lowry´s method and data are expressed as pmol/μg of protein.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!