ATP was measured with a ATP determination kit (Molecular Probes), following the instructions of the manufacturer28 (link). Cells were lysed with 1× Passive Lysis Buffer (Promega) and were diluted 1:10 using the ATP determination kit reactant mix leading to a total volume of 100 μl. Luminescence was determined directly after the addition of the lysate to the reaction, and was quantified in a Luminometer (Turner Designs, Sunnyvale, CA). ATP concentrations in experimental samples were calculated by using an ATP standard curve.
Atp determination kit
Manufactured by Promega
Sourced in United States
The ATP determination kit is a laboratory tool used to measure the concentration of adenosine triphosphate (ATP) in various samples. ATP is a crucial energy-carrying molecule found in all living cells. The kit provides reagents and protocols to quantify ATP levels, which can be used to assess cellular activity and energy metabolism.
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Lab products found in correlation
Atp determination kit, by Thermo Fisher Scientific (2 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Isoflurane, by Merck Group (1 mentions)
Goat anti rabbit igg h l fitc, by Abcam (1 mentions)
Rt qpcr kit, by Takara Bio (1 mentions)
Trizol, by Omega Bio-Tek (1 mentions)
Mts assay kit, by Promega (1 mentions)
Sb431542, by Merck Group (1 mentions)
Glomax luminometer, by Promega (1 mentions)
Alexa fluor 488 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti calreticulin antibody, by Abcam (1 mentions)
Varioscan flash, by Thermo Fisher Scientific (1 mentions)
Varioskan flash, by Thermo Fisher Scientific (1 mentions)
6 protocols using atp determination kit
1
Quantifying Cellular ATP Levels
2
Chondrocyte Differentiation Protocol
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d -Glucosamine HCl (purity > 98.5 %, MB1695) was purchased from Dalian Meilun Biotechnology™ (Dalian, China). Dexamethasone (No. D1756) and isoflurane were acquired from Sigma-Aldrich (St. Louis, MO, USA) and Baxter Healthcare™ (Deerfield, IL, USA), respectively. Primary antibody dilution buffer (NO. G2025) was procured through Servicebio ™ (Wuhan, China). Trizol® was procured through Omega Bio-Tek™ (Doraville, GA, USA). The SYBR Green dye and reverse transcription and real-time quantitative polymerase chain reaction (RT-qPCR) kits were procured through TaKaRa Biotechnology™ (Dalian, China). Phospho-SMAD Family Member 2 (p-Smad2) (AP0548) and ACAN (A8536) were procured through Abclonal™ (Wuhan, China); TGFβR1 (ab31013), COL2A1 (ab34712), Smad2 (ab40855), SOX9 (ab185230) antibodies and goat anti-rabbit IgG H&L (FITC) (ab6717) were procured through ABCAM™ (Shanghai, China). Oligonucleotide primers were procured through TIANYIHUIYUAN™ (Guangzhou, China). DMEM/F-12 medium and fetal bovine serums (FBS) were procured through Gibco™ (Carlsbad, California, USA). SB431542 was procured through Merck ™ (Beijing, China). MTS Assay Kit® and ATP Determination Kit® (S0026) were procured through Promega™ (Fitchburg, Wisconsin, USA) and Beyotime™ (Wuhan, China), separately. The remaining materials consisted of analytical-grade reagents/chemicals.
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3
Quantifying Cellular ATP Levels
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Cellular ATP was quantified using an ATP determination kit according to the manufacturer's instructions (Promega, San Luis Obispo, CA, USA). Briefly, cells, which had been plated at equal densities, were lysed in passive lysis buffer. Equal volumes of cell lysate were then added to the standard reaction solution, and luminescence was measured and normalized to the protein amount of each lysate.
4
Quantifying Cellular ATP Levels
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ATP was assessed using ATP determination kit (Invitrogen) according to manufacturer instruction. HUVECs were harvested and counted, then lysed in H2O (1 ml/1 × 106 cells) and boiled for 10 min. After spinning, the supernatants were assessed with the ATP determination kit, and luciferase activity was measured by Glomax luminometer (Promega, Madison, WI).
5
Calreticulin Exposure and ATP Release
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Calreticulin (CRT) exposure. LL/2 and MC-38 cells were seeded in duplicate onto 6 well plates at 5×105 cells/well. LL/2 cells were treated in triplicates with EV-LL/2 (10 particles/cell), EV-MC-38 (10 particles/cell), Virus (10vp/cell), EV-Virus-LL/2 (10 particles/cell), EV-Virus-MC-38 (10 particles/cell). MC-38 cells were treated in triplicates with EV-MC-38 (10 particles/cell), EV-LL/2 (10 particles/cell), Virus (10vp/cell), EV-Virus-MC-38 (10 particles/cell), EV-Virus-LL/2 (10 particles/cell). After 24 h cells were harvested and stained with 1:1000 diluted rabbit polyclonal anti-Calreticulin antibody (Abcam, Cambridge, UK) for 40 min at 4°C subsequently with 1:100 diluted Alexa-Fluor 488 secondary antibody (Invitrogen, Carlsbad, CA) and analyzed by flow cytometry (LSR II, BD, Franklin Lakes, NJ).
ATP release. LL/2 and MC-38 cell lines were seeded in triplicates onto 96 well plates at 1×104 cells/well and treated as mentioned above. Supernatants were collected after 48 h and analyzed with ATP Determination Kit according to manufacturer's protocol (Promega, Madison, WI) for luminometric analysis (Varioscan Flash, ThermoFisher Scientific, Waltham, MA).
ATP release. LL/2 and MC-38 cell lines were seeded in triplicates onto 96 well plates at 1×104 cells/well and treated as mentioned above. Supernatants were collected after 48 h and analyzed with ATP Determination Kit according to manufacturer's protocol (Promega, Madison, WI) for luminometric analysis (Varioscan Flash, ThermoFisher Scientific, Waltham, MA).
6
Quantifying ATP Release from Infected Cells
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Forty-eight hours after cell infection, supernatants were collected and analyzed with the ATP Determination Kit according to the manufacturer’s protocol (Promega, Madison, WI, USA) by luminometric analysis (Varioskan Flash, ThermoFisher Scientific, Waltham, MA, USA). Untreated cells were used as control. The percentage of released ATP was calculated according to the following equation:
where RLU is the relative light unit.
where RLU is the relative light unit.
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