Atp assay kit

Following the culture period, cells were transferred to 2.5 mM glucose in SAB buffer for 2 h, followed by transfer to either 2.5 mM glucose SAB buffer or 16.7 mM glucose SAB buffer for 1 h. Cells were washed with PBS, harvested by trypsinisation and pelleted by centrifugation. The cells were lysed in 150 μL 1M perchloric acid on ice to precipitate cellular proteins. Lysate was centrifuged at 20,000× g for 10 min, after which 150 μL supernatant was transferred to a new tube with 150 μL 1M KOH. ATP was quantified using the ATP assay kit (Life Technologies, Carlsbad, CA, USA).

The intracellular and extracellular ATP concentrations of S. aureus were determined according to the method described previously with some modifications.^{4 (link)} Briefly, logarithmic phase S. aureus cells were harvested by centrifugation at 3000 rpm for 5 min, washed twice and resuspended in 0.85% sterile saline at a cell density of 1 × 10⁹ CFU mL⁻¹. The bacterial suspensions were treated with different concentrations of CHQA at 37 °C for 30 min. Then, the samples were centrifuged at 5000 rpm for 5 min, the supernatants and cell pellets were stored on ice respectively to prevent ATP loss. The ATP concentration of the supernatants, which represents the extracellular concentration, was determined using an ATP assay kit (Life Technologies, Eugene, OR, USA) following manual instructions with a microplate reader (PE envision, Perkin Elmer Co., Waltham, MA, USA). To measure the intracellular ATP concentration, the cell pellets were washed, resuspended in 0.85% sterile saline, and treated by ultrasound on ice. After centrifugation at 10 000 rpm for 3 min, the ATP concentration of the resultant supernatants, which represents the intracellular concentration, was determined as described for the extracellular ATP concentration.

Enzymatic activity assays were performed on larval whole cell extracts or isolated mitochondria from third instar larvae as previously described [13 (link),116 (link)]. Polarography was performed on isolated mitochondria from third instar larvae as previously described [13 (link),117 (link)]. Aconitase activity assays were performed in isolated mitochondria from third instar larvae as previously described [13 (link)]. ATP level for larvae, eyes, and heads were determined by ATP assay kit (Invitrogen) [118 (link),119 (link)]. Flies were exposed to light (~1,800 Lux) for 1 h prior to detection of ATP levels in adult eyes and heads. Eyes were dissected in PBS, and heads were frozen on dry ice and separated on a metal plate kept on dry ice. Five third instar larvae, 20 eyes or 5 heads were dissected and homogenized in 50 μl of 100 mM Tris and 4 mM, EDTA, pH 7.8. These homogenates were snap-frozen in liquid nitrogen and then boiled for 3 min. Samples were then centrifuged, and the supernatant was diluted (1/50 for larvae and 1/2 for heads and eyes) in extraction buffer mixed with luminescent solution. Luminescence was measure on FLUOstar OPTIMA plate reader. DHE staining was performed as described previously [78 (link)]. Flies were exposed to 24 h light (1,800 Lux) prior to DHE staining in adult eyes.

ATP assays were performed on L4 and L5 leaves isolated from four biological replicates of WT and P07 plants. ATP extraction was performed as previously described [70 (link)], with minor modifications. The leaves were ground in liquid nitrogen, and 30 mg of leaf tissue was homogenized with 300 μL of 0.1 M HCl for 5 min. The homogenate was centrifuged at 20,000 × g for 10 min, and the supernatant was centrifuged using a microconcentrator Ultra-15 (Millipore, Billerica-USA) at 14,000 × g for 20 min. The ATP content was determined by using an ATP assay kit (Invitrogen) and a GloMax luminometer (Promega, Fitchburg, WI, USA).

ATP content was determined using the ATP assay kit (Invitrogen) essentially as described [10 (link)], with minor modifications. Entire 3-week-old seedlings from WT and OE lines were ground in liquid nitrogen, and 200 mg of tissue powder homogenized in a 4:2:1 methanol/chloroform/water mixture (1 ml). Samples were vortexed, kept on ice for 30 min and vigorously homogenized for additional 15 min. After the addition of water (1 ml), the samples were centrifuged at 12.200 x g for 5 min for phase separation and subsequently dried in a centrifugal vacuum concentrator. The pellet was resuspended in 10 mM phosphate buffer (pH 7.4). Measurement of ATP content was carried out on a GloMax-Multi Detection System (Promega). The obtained data were subjected to ANOVA (t test), and considered significantly different at the P < 0.05 probability level.