Aposensor cell viability assay kit

Preosteoblast MC3T3-E1 cells (CRL-2593; ATCC, Manassas, VA) were cultured as described previously^{25 (link)}. Briefly, cells were cultured in complete alpha minimal essential media (α-MEM) containing 10% fetal bovine serum (FBS) (Invitrogen and Atlanta Biologicals, Atlanta, GA) and 1% Penicillin-Streptomycin (Life Technologies). Passages between 18 and 24 were used for the experiments. For gene expression analysis, cells were plated in 96-well plates (@ 20,000 cells/well) (Corning Incorporated, Corning NY). Following 24 hours of plating in complete α-MEM, cells were treated as indicated for 6 hours. RNA extraction was completed as described below.
For assessment of intracellular ATP, MC3T3-E1 cells were plated @10,000 cells/well in 96-well plates (white-walled from Corning Incorporated, Corning NY). Twenty four hours after plating, cells were treated as indicated for 6 hours. Intracellular ATP levels were assessed using the ApoSensor Cell Viability Assay kit (BioVision, San Francisco, CA) and Luminescence was measured by Synergy/neo2 multi-mode plate reader and calculated with Gen5 software (Bio-Tek). Each experiment was done in duplicates.

An ATP Assay was performed for determining the viability of the coral oocytes. The productivity of the mitochondria, as indicated by the number of ATPs, indicated the degree of harm for the oocytes in a treatment. Coral oocytes were placed in a test tube, and seawater was drawn using a pipette. Oocyte viability was assessed through a luminescence assay (ApoSENSOR Cell Viability Assay Kit, BioVision, Cambridge BioScience, UK). In brief, a nucleotide-releasing buffer (100 μL) was added to the test tube, and after 3–5 min, an ATP-monitoring enzyme (5 μL) was added. After approximately 30 s to 1 min, the test tube was placed into a luminometer (Lumat LB 9507, Berthold Technologies, Germany), and readings were taken27 (link).

ATP bioassay was conducted prior to and after Breviolum cryopreservation to compare the change in Breviolum viability throughout the experiment. In normal cells, ATP acts as the energy currency for cellular functions, whereas its concentration reduces when the cell dies. Hence, ATP bioassays can be used as a preliminary assessment of cellular survival rate. This study performed ATP bioassays using ApoSENSOR™ Cell viability assay kit (BioVision, Milpitas, CA, USA). First, 5 μL of the sample was put into a luminescence test tube, followed by adding 100 μL of nucleotide releasing buffer, and was left for 3 min at room temperature for completion of the reaction. This was followed by adding 5 μL of ATP monitoring enzyme, thorough mixing and rest period another 30 s at room temperature. Finally, a measurement was made using a luminometer (Lumat LB 9507, Berthold Technologies GmbH & Co. KG, Germany). The use of nucleotide releasing buffer ruptured the Breviolum, allowing the binding of luciferin to ATP, catalyzed by luciferase, and the oxidation of ATP into adenosine monophosphate (AMP) and two phosphate groups, emitting a light blue light. The light was captured and measured by the luminometer for ATP concentration based on the light intensity.

Intracellular ATP levels were measured using the ApoSENSOR cell viability assay kit (BioVision) according to the manufacturer’s instructions. Briefly, cells were treated with DHM (10, 50 and 100 μM) for 48 h, then incubated with 100 μl nuclear releasing reagent for 5 min at room temperature with gentle shaking, followed by further incubation with 5 μl ATP monitoring enzyme. Detection was performed using a luminometer (Sirius L; Titertek-Berthold, Pforzheim, Germany).