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This protocol describes fluorescence imaging and analysis of the endogenous metabolic coenzymes, reduced nicotinamide adenine (phosphate) dinucleotide (NAD(P)H), and oxidized flavin adenine dinucleotide (FAD). Autofluorescence imaging of NAD(P)H and FAD provides a label-free, nondestructive method to assess cellular metabolism.
Cellular metabolism is the process by which cells generate energy, and many diseases, including cancer, are characterized by abnormal metabolism. Reduced nicotinamide adenine (phosphate) dinucleotide (NAD(P)H) and oxidized flavin adenine dinucleotide (FAD) are coenzymes of metabolic reactions. NAD(P)H and FAD exhibit autofluorescence and can be spectrally isolated by excitation and emission wavelengths. Both coenzymes, NAD(P)H and FAD, can exist in either a free or protein-bound configuration, each of which has a distinct fluorescence lifetime-the time for which the fluorophore remains in the excited state. Fluorescence lifetime imaging (FLIM) allows quantification of the fluorescence intensity and lifetimes of NAD(P)H and FAD for label-free analysis of cellular metabolism. Fluorescence intensity and lifetime microscopes can be optimized for imaging NAD(P)H and FAD by selecting the appropriate excitation and emission wavelengths. Metabolic perturbations by cyanide verify autofluorescence imaging protocols to detect metabolic changes within cells. This article will demonstrate the technique of autofluorescence imaging of NAD(P)H and FAD for measuring cellular metabolism.
Metabolism is the cellular process of producing energy. Cellular metabolism encompasses multiple pathways, including glycolysis, oxidative phosphorylation, and glutaminolysis. Healthy cells use these metabolic pathways to generate energy for proliferation and function, such as the production of cytokines by immune cells. Many diseases, including metabolic disorders, cancer, and neurodegeneration, are characterized by altered cellular metabolism1. For example, some cancer cell types have elevated rates of glycolysis, even in the presence of oxygen, to generate molecules for the synthesis of nucleic acids, proteins, and lipids2<....
1. Cell plating for imaging
The epithelial breast cancer cell line, MCF-7, was cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. For fluorescence imaging, the cells were seeded at a density of 4 × 105 cells per 35 mm glass-bottom imaging dish 48 h before imaging. The cells were imaged before and after cyanide treatment using the protocols stated above. The goal of the cyanide experiment is to confirm spectral isolation of NAD(P)H and FAD fluorescence and validate the imaging system and a.......
Autofluorescence intensity and lifetime imaging have been widely used to assess metabolism in cells21,55. FLIM is high resolution and therefore resolves single cells, which is important for cancer studies because cellular heterogeneity contributes to tumor aggression and drug resistance7,39,41,44,45
Funding sources include the Cancer Prevention and Research Institute of Texas (CPRIT RP200668) and Texas A&M University. Figure 1Â was created with BioRender.com.
....Name | Company | Catalog Number | Comments |
2-deoxy-d-glucose (2-DG) | Sigma | AC111980000; AC111980010; AC111980050; AC111980250 | |
Antibiotic Antimicrobial (pen-strep) | Gibco | 15240096 | |
Cell Samples | American Type Culture Collection | N/A | MCF-7 cancer line |
CellProfiler | Broad Institute | N/A | Image analysis software |
Conical Tube | VWR | 89039-664 | 15 mL conical tube |
DMEM | ThermoFisher | 11965092 | Culture media |
FAD dichroic mirror | Semrock | FF495-Di03-25x36 | 495 nm |
FAD emission filter | Semrock | FF01-550/88-25 | 550/88 nm |
FAD excitation filter | Semrock | FF01-458/64-25 | 458/64 nm |
FBS | ThermoFisher | 16000036 | |
Fluorescence Lifetime Microscope | 3i | N/A | |
Glass bottom dish | MatTek Corp | P35G-1.0-14-C | |
Multiphoton Laser | Coherent | N/A | 2P Coherent Laser, Tunable 680 nm-1080 nm |
NAD(P)H dichroic mirror | Semrock | FF409-Di03-25x36 | 409 nm |
NAD(P)H emission filter | Semrock | FF02-447/60-25 | 447/60 nm |
NAD(P)H excitation filter | Semrock | FF01-357/44-25 | 357/44 nm |
PBS | ThermoFisher | 70011044 | |
Potassium Cyanide | Sigma-Aldrich | 380970 | |
SlideBooks 6 | 3i | N/A | Image acquisition software |
SPCImage | Becker & Hickl GmbH | N/A | Fluorescence lifetime analysis software |
Stage Top Incubator | okoLab | N/A | |
Trypsin | Biosciences | 786-262 | |
Urea | Sigma-Aldrich | U5128 | |
YG beads | Polysciences | 19096-2 | Yg microspheres (20.0 µm) |
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