Yes, NAD/NADH can be determined directly in 96-well plate. Please see our optimized protocol Direct NAD/NADH Assays in 96-Well Plate
If direct sample processing is not possible, we recommend snap freezing tissue samples in liquid nitrogen and keeping them either in liquid nitrogen or at -80°C until further processing.
The second method is better because the first method may cause mitochondrial fragmentation and thus may alter intracellular NAD/NADH concentrations. The second method involves less pretreatment. Adding the heated extraction buffer terminates the metabolic reactions in the cells and is thus much less likely to alter the intracellular NAD/NADH concentrations.
We have not done these experiments. Your results are very interesting, and not unexpected. In general, cells are healthy at certain densities. When cells lack contacts for a healthy environment at low density, they are likely to produce less NAD/NADH, and if too dense, the cells become less active and thus would reach a plateau in NAD/NADH concentrations.
Chaurasiya, A., et al. (2021). Pathogen induced subversion of NAD+ metabolism mediating host cell death: a target for development of chemotherapeutics. Cell Death Discovery 7(1): 10. Assay: NAD and NADH in microbial cells.
Joe, Y., et al. (2020). Cross-talk between CD38 and TTP is essential for resolution of inflammation during microbial sepsis. Cell Reports 30(4): 1063-1076.e5. Assay: NAD and NADH in mouse cells.
Zhang, M., et al. (2020). Dysregulated metabolic pathways in age-related macular degeneration. Scientific Reports 10(1): 2464. Assay: NAD and NADH in human cells.
Liemburg-Apers, Dania C., et al (2016). Acute stimulation of glucose influx upon mitoenergetic dysfunction requires LKB1, AMPK, Sirt2 and mTOR-RAPTOR. J Cell Sci 129.23: 4411-4423. Assay: NAD/NADH in mouse cells.
Kim, Ha-Neui, et al. (2015) Sirtuin1 suppresses osteoclastogenesis by deacetylating FoxOs. Molecular Endocrinology 29.10: 1498-1509. Assay: NAD/NADH in mice.
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