Cells were centrifuged at 10,000× g for 5 min at 4 °C. The pellets were treated with a lysis buffer from an ATP detection kit (Beyotime, Haimen, China) for 1 min at room temperature and then were centrifuged at 10,000× g for 5 min. The supernatant was transferred to a new 1.5 mL tube for an ATP test with the ATP detection kit purchased from Beyotime (China). The relative ATP content was determined using the formula: relative ATP content = ATP value/protein value. Protein concentration in the sample was quantified using a Bradford 1× Dye Reagent (Bio-Rad, Hercules, CA, USA), measured at a wavelength of 595 nm.
Atp detection kit
Manufactured by Beyotime
Sourced in China
The ATP detection kit is a laboratory tool designed to quantify the levels of adenosine triphosphate (ATP) in a sample. ATP is a fundamental energy-carrying molecule found in all living cells. The kit provides the necessary reagents and protocols to accurately measure ATP concentrations, enabling researchers to study cellular metabolism, energy production, and other biological processes.
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Lab products found in correlation
Infinite 200 pro, by Tecan (5 mentions)
Mitotracker, by Thermo Fisher Scientific (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Lactic acid assay kit, by Nanjing Jiancheng (2 mentions)
Arl67156, by Merck Group (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Hoechst 33258, by Beyotime (2 mentions)
Glomax 20 20, by Promega (2 mentions)
Infinite m200 pro, by Tecan (2 mentions)
Bicinchoninic acid assay, by Beyotime (2 mentions)
Adi eks 700b, by Enzo Life Sciences (1 mentions)
Hmgb1 elisa kit, by Arigo Biolaboratories (1 mentions)
Prism 9, by GraphPad (1 mentions)
Uv 1800pc, by MAPADA (1 mentions)
Bca protein assay reagent kit, by Beyotime (1 mentions)
Victor2 multilabel counter, by PerkinElmer (1 mentions)
Er tracker, by Thermo Fisher Scientific (1 mentions)
Rpim 1640 medium, by Thermo Fisher Scientific (1 mentions)
Mitosox detection kit, by Thermo Fisher Scientific (1 mentions)
107 protocols using atp detection kit
1
Quantifying Cellular ATP Levels
2
ATP Quantification in Bacterial Cells
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The ATP concentration was determined using the ATP detection kit (Biyuntian (Shanghai, China), S0027). BBR, TA, BBT/TA MIX, and BBR-TA NPs were added to the logarithmic phase bacterial suspension, and the final concentrations of 62.50, 31.25, and 15.63 μg/mL were achieved. After incubation at 37 °C for 3 h, bacterial cells were collected, washed, and treated with 0.6 mL of ice-cold lysis buffer to extract ATP. The bacterial solution was centrifuged at 4500 rpm for 10 min. The top layer was collected and frozen to prevent ATP loss. The supernatant and the same amount of ATP were mixed in a black opaque 96-well microtitration plate and measured using the multifunctional microplate detector (Tecan spark, Tecan, Männedorf, Switzerland).
3
Quantification of CRT, ATP, HMGB1, and HSP70/90
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CRT: Cells were collected with 0.25% trypsin without EDTA, washed twice with cold PBS, and incubated with Alexa Fluor 488 fluorescent anti-calreticulin antibodies in 100 μl of FACS buffer at a 1:100 dilution at 4 °C in darkness for 1 h. The expression of calreticulin (CRT) was analyzed by flow cytometry. ATP: The working fluid was configured according to the instructions of the ATP detection kit (#S0026, Biyuntian). HMGB1: According to the instructions of the high mobility group box-1 protein (HMGB1) ELISA kit (#ARG81351, Arigo), the supernatant of the cells was collected and assessed. HSP70/90: According to the instructions of the HSP70/90 ELISA kit (ADI-EKS-700B, ENZO), the supernatant of the cells to be tested was collected and assessed.
4
Metabolic Profiling of OC Cells
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Glucose uptake and lactate production kits (Nanjing Jiancheng, Nanjing, China) and an ATP detection kit (Biyuntian, China) was used to detect glucose, lactate, and ATP, respectively. OC cells were seeded into 6-well plates. We collected the cells and culture solution after treatment. Finally, we tested them according to the operating instructions.
5
Quantifying Cellular ATP and Mitochondrial Pore Activity
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ATP detection kit (Biyuntian, Shanghai, China) was used to detect ATP concentration. In brief, after hypoxia-reoxygenation, the cells were fully lysed by adding 200 μl of cell lysis and then centrifuged for 5 min at 4 °C and 12,000 g to remove insoluble substances. Then the supernatant was collected and incubated with the ATP working solution. Relative light units (RLU) were measured by chemiluminometer (Varioskan lux, Thermol Biotech, USA). The ATP productions were calculated on the basis of the standard curve and expressed as nmol/mg protein.
mPTP opening was tested by an mPTP detection kit in line with the manufacturer’s instructions. In short, after washing with PBS, the AML12 cells were cultured with calcein-AM/CoCl2 working buffer for 30 min at 37 °C. Subsequently, the buffer was removed and replaced with prewarmed fresh medium, and then the cells were further incubated for 30 min at 37 °C. Afterward, the cells were washed 2–3 times with PBS, and the fluorescence was viewed by an inverted fluorescence microscope (Olympus IX71, Japan).
mPTP opening was tested by an mPTP detection kit in line with the manufacturer’s instructions. In short, after washing with PBS, the AML12 cells were cultured with calcein-AM/CoCl2 working buffer for 30 min at 37 °C. Subsequently, the buffer was removed and replaced with prewarmed fresh medium, and then the cells were further incubated for 30 min at 37 °C. Afterward, the cells were washed 2–3 times with PBS, and the fluorescence was viewed by an inverted fluorescence microscope (Olympus IX71, Japan).
6
Quantifying Cellular ATP Levels
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The ATP level of cells was measured with ATP detection kit (Biyuntian Biotechnology Co., Ltd. China).
The operation steps were as follows: 1 × 10 7 cells was collected and put into centrifuge tube. 200 µl ATP cracking liquid was added to ensure that cells were completely lysed. Centrifugation of 12000 g at 4 ℃ for 5 minutes, collect the supernatant. The protein concentration was determined by BCA method, and the samples were adjusted to an uniform concentration with ATP detection lysate, so as to eliminate the in uence of different protein concentration. Add ATP detection working solution in 96 well plate according to 100 µl/well, put it at room temperature for 5 minutes to consume background value. Then add 20 µl standard product or tested sample to each hole respectively, and mixed. Luminometer function of full band enzyme scale was used to measure relative light unit (RLU). According to the RLU value of the standard product, the standard curve was established, and the ATP contents of the tested samples were calculated. The results of each group were statistically analyzed with the percentage of the control group.
The operation steps were as follows: 1 × 10 7 cells was collected and put into centrifuge tube. 200 µl ATP cracking liquid was added to ensure that cells were completely lysed. Centrifugation of 12000 g at 4 ℃ for 5 minutes, collect the supernatant. The protein concentration was determined by BCA method, and the samples were adjusted to an uniform concentration with ATP detection lysate, so as to eliminate the in uence of different protein concentration. Add ATP detection working solution in 96 well plate according to 100 µl/well, put it at room temperature for 5 minutes to consume background value. Then add 20 µl standard product or tested sample to each hole respectively, and mixed. Luminometer function of full band enzyme scale was used to measure relative light unit (RLU). According to the RLU value of the standard product, the standard curve was established, and the ATP contents of the tested samples were calculated. The results of each group were statistically analyzed with the percentage of the control group.
7
Intracellular Metabolic Changes in IDH2 Mutant HEK293T Cells
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HEK293T cells were lysed 48 h post-transfection with IDH2 WT and mutant constructs under normal and/or hypoxic conditions (200 µM CoCl2 treatment). Changes in intracellular lactic acid and glucose levels were determined with the Lactic Acid Assay Kit (Jiancheng Bioengineering Institute, Nanjing), the Glucose Assay Kit (Rongsheng Biological Pharmaceutical Co. Shanghai), and the ATP Detection Kit (Beyotime Biotechnology Co. Shanghai). According to the manufacturer`s instructions, the changes in the absorption value at 505 nm (glucose) or 530 nm (lactic acid) were measured by UV spectrophotometry (UV-1800PC, MAPADA) after the lysate proteins (5.2 μg/μL) were added to the working solution. ATP levels were measured by Luminometer (Lumat LB 9507, Berthold technologies) after the lysate proteins (2 μg/μL) were added to the working solution. All assays were performed in triplicate. The data were finally analyzed and exported by GraphPad Prism 9.0.0 (GraphPad Software, LLC).
8
CSLF3872 Modulates Extracellular ATP in Bacteria
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The indicator bacteria (S. aureus, E. coli, Salmonella, and Campylobacter) in the logarithmic phase were centrifuged and resuspended in PBS (pH 7.0) with OD600 = 1.0 (4 – 5 × 108 CFU/mL). Bacteria were treated for 2.5 h at 37 °C in the presence of CSLF3872 (100µg/mL), and extracellular ATP levels after CSLF3872 treatment were detected by an ATP detection kit (Beyotime, Shanghai, China). Luminescence detection was performed using an Infinite 200 PRO microplate reader (Tecan, Männedorf, Switzerland).
9
ATP Quantification by Luminometric Assay
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The amount of ATP was measured by the ATP detection kit (Beyotime, China). CCs were lysed on ice with 100 μL lysis buffer from ATP detection kit. After being centrifuged at 12,000 g for 5 min at 4°C, the supernatant was transferred to a new 1.5 mL tube for ATP test. Protein concentrations were determined by using a BCA Protein Assay Reagent Kit (Beyotime, China). The luminescence from a 100 μL sample was assayed in a luminometer together with 100 μL of ATP detection buffer from the ATP detection kit. The standard curve of ATP concentration was prepared from a known amount (1 nM–10 μM).
10
Quantitative ATP Measurement in HL-1 Cells
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The ATP concentration was detected using an ATP detection kit (Beyotime). In simple terms, HL-1 cells (1×105/well) were collected by trypsinization, followed by centrifugation and washing with PBS. The cells were mixed with RIPA lysis buffer containing protease inhibitors at 4°C for 10 min, and then centrifuged at 4°C for 5 min at 12,000 × g. Subsequently, the cell supernatant was incubated with 300 µL of kit solution for 5 min, and the ATP level in the supernatant was measured via enzyme labeling using a luminometer to detect luminescence produced by the luciferase reaction in the ATP detection kit. Triplicates were performed for each sample to ensure accuracy and reproducibility of the results.
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