Cell Meter™ Intracellular NADH/NADPH Flow Cytometric Analysis Kit *Deep Red Fluorescence*

Protocol summary

1. Prepare cells (0.5 - 1×10⁶ cells/mL)
2. Incubate cells with test compounds and JJ1902 NAD(P)H Sensor at 37 ^oC for 20-30 minutes
3. Wash and keep cells in Assay Buffer
4. Analyze cells with a flow cytometer using APC channel

Important
Thaw all the kits components at room temperature before starting the experiment.

1. For each sample, prepare cells in 0.5 mL serum-free medium or buffer of your choice at a density of 1×10⁵ to 1×10⁶ cells/mL. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density. For adherent cells, gently lift the cells with 0.5 mM EDTA to keep the cells intact, and wash the cells once with serum-free media. Note: JJ1902 NAD(P)H sensor is compatible in the presence of serum as well. The optimization of the conditions for the sensor is highly recommended cell line to cell line.
                                                                                                                                                                                         
2. Incubate cells with test compounds at 37 ^oC for a desired period of time to stimulate intracellular NADH/NADPH. Note: The appropriate incubation time depends on the individual cell type and test compound used. Optimize the incubation time for each experiment.
   
   
3. Add 1 µL of JJ1902 NAD(P)H Sensor (Component A) into 0.5 mL cell suspension. Incubate at 37 ^oC for 30-60 minutes. Note: For a NADH/NADPH positive control treatment: Jurkat cells were incubated with 100 µM NADH or NADPH for 30 minutes in serum-free medium, and co-incubated with JJ1902 NAD(P)H Sensor working solution at 37 ^oC for another 30 minutes. See Figure 1 for details.
   
   
4. Wash cells with your desired buffer such as HHBS or DPBS once. Keep cells in Assay Buffer (Component B).
   
   
5. Monitor the fluorescence intensity at APC channel using a flow cytometer. Gate on the cells of interest, excluding debris.