Atp bioluminescence kit cls 2

The 1 × 10⁶ promastigotes (WT, LdU^−/+, LdU^−/− and LdU^−/−AB) were resuspended in reaction buffer containing 1 mM dithiothreitol, 0.5 mM luciferin, and 12.5 µg/ml luciferase and mixed gently, after which readings were taken [37 (link)]. ATP standard curve was prepared run in all experiments with different concentrations of ATP, calculations were made against the curve and cellular ATP levels were expressed as nmol/10⁶ cells.
In case of amastigotes, a crude mitochondria preparation from WT, LdU^−/+ and LdU^−/+AB amastigotes was obtained as described elsewhere [38 (link)]. ATP production was measured in the presence of indicated substrates (succinate, pyruvate and α-ketoglutarate) and 67 μM ADP. Inhibitors (6.7 mM malonate, 33 μg/ml attractyloside) were pre-incubated with mitochondria for 10 min on ice. The concentration of ATP was determined by Luminometer using the ATP Bioluminescence kit CLS II (Roche Applied Science, IN).

ATP content was measured using the ATP Bioluminescence Kit CLS II (Roche). Immediately after dissection, single retinas were incubated in 50 μl of 1% perchloric acid for precisely 10 min at room temperature before the reaction was stopped in 450 μl of boiling Tris buffer (100 mM Tris, 4 mM EDTA, pH 7.75), incubated for 2 min at 100°C, and centrifuged at 1000 g for 1 min. For further details, see supplementary Materials and Methods.

For ATP and mitochondrial membrane potential analyses, cells were grown on 24-well plates. For ATP quantification, cells were lysed on ice in TE buffer (100 mM Tris +4 mM EDTA, pH = 7.5), scraped, boiled, and centrifuged at 18,000×g. ATP in supernatant was measured using ATP Bioluminescence Kit CLS II (Roche). Membrane potential was determined using JC-1 dye (Molecular Probes) as described [31] (link). Fluorescence data from microplate reader were expressed as ratios of aggregate to monomer.

Determination of ATP level was performed using the ATP Bioluminescence CLS II kit following manual instruction (Roche, lifescience.roche.com). Leydig cells (2 × 10⁶/0.5 mL) were incubated in shaking-water bath (1 h/34 °C/80 cycles per min) and centrifuged (1200× g/5 min). Precipitated cells were resuspended in boiling water and Tris-Ethylenediaminetetraacetic acid (EDTA) (1:9), boiled for 2 more minutes, centrifuged (900× g/1 min) and final supernatant was used for ATP measurement. Sample/standard and Luciferase reagent were mixed 1:1 and luminescence was measured by the Biosystems/luminometar (Fluoroscan, Ascent, FL, USA). Mitochondrial abundance and mitochondrial membrane potential (ΔΨm) were determined by mitotrack green and tetramethylrhodamine (TMRE), respectively, staining for 20 min/34 °C/5% CO₂ were applied with subsequent fluorescence reading (Fluoroscan, Ascent, FL, USA). The excitation wavelength used for the each test was 485 and 550 nm while emission wavelengths were 510 and 590 nm respectively; after staining, cells were washed with 0.1% BSA-PBS [31 (link),33 (link),36 (link)]. Oxygen consumption by Leydig cells suspension was measured by Clark electrode at 34 °C and oxygen uptake and oxygraphic curves were obtained by digital multimeter VC820 (Conard Electronic, Hirsau, Bavaria, Germany) and software Digiscope for Windows (version 2.06) [31 (link)].

Escherichia coli MET1158 strain, E. coli LW7 pLW11 and E. coli pBAC-LacZ were donated by Prof Karina Xavier (Instituto Gulbenkian de Ciência, Portugal), Prof. William Bentley (University of Meriland, USA) and Keith Joung (Addgene plasmid # 13422), respectively.
All chemicals were purchased from Sigma (USA) if not otherwise stated. DPD was acquired from Carbosynth (Compton, Berkshire, UK). PD-10 desalting columns and Protino^® Ni-NTA columns (1 mL) for protein purification were purchased from GE Healthcare Lifescience (Chicago, IL, USA) and Macherey-Nagel (Düren, Germany), respectively. Pierce Coomassie Plus Assay Kit was obtained from Thermo Fisher Scientific (Waltham, MA, USA). ATP Bioluminescence CLS II kit was purchased from Roche Diagnostics GmbH (Basel, Switzerland), Kinase-Glo Max Luminescent kinase assay kit from Promega Corp. (Madison, WI, USA) and ADP-Quest kit from DiscoveRx Corp. (Fremont, CA, USA). PopCulture™reagent and rLysozyme™ were purchased from Millipore (Burlington, MA, USA). Plates were purchased from Greiner Bio One (KremsMünster, Austria), for assay development and screening campaign, and from Thermo Fisher Scientific (Waltham, MA, USA) for AI-2 QS interference assay.

The ATP level was determined using the ATP Bioluminescence CLS II kit following the manual instruction (Roche Diagnostics, Indianapolis, USA) published previously by our group (15 (link)). Leydig cells (1 × 10⁶/tube) were resuspended in boiling water and Tris-EDTA (1:9), incubated in the water bath (100°C/3 min), centrifuged (900 × g/1 min), and the supernatant was used for ATP measurement while cell pellet was further used for Bradford method analysis. Sample/standard and Luciferase reagent were mixed 1:1, and luminescence was measured by the Biosystems/luminometer (Fluoroscan, Ascent, FL). For ΔΨm detection, as we described before (15 (link), 22 (link)), Leydig cells were placed in 96 well-plates (1 × 10⁵ cells/well) and incubated with tetramethylrhodamine (TMRE) staining for 20 min/34°C/5%CO₂. Fluorescence was measured on fluorimeter (Fluoroscan, Ascent, FL) on excitation wavelengths 485 and 550 nm, while emission wavelengths were 510 and 590 nm. Cells were washed with 0.1%BSA–PBS and stored for protein quantification by Bradford method.