Live Cell Imaging with BrightCell™ Photostable Cell Culture Media

Protection from Light-Induced Cellular Damage

Fluorescent detection of targets in live cultured cells, including fluorescence microscopy, optogenetics, and fluorescence activated cell sorting (FACS) often require prolonged exposure to high levels of blue light. The wide use of green fluorescent protein (GFP) as a fluorescent protein tag in live cell imaging requires blue light (470 nm) excitation. Also, recently discovered optogenetic actuators such as channelrhodopsin-2 (ChR2), melanopsin (OPN4), cryptochrome-2 (Cry2), and light-oxygen-voltage sensitive proteins (LOV) universally depend on light in the blue range of the visible spectrum for their photoactivation. However, cell culture media and supplements often contain components that are converted to toxic free radicals by exposure to light of certain wavelengths. In particular, DMEM (Dulbecco’s Modified Eagle Medium) and Neurobasal^® medium, as well as culture media supplements such as B-27, N2, SATO and NS21, can lead to marked disruption of cellular metabolism and significant increases in cytotoxicity when exposed to blue light.

BrightCell™ Photostable Media products are cell culture media and supplements that have been reformulated with distinct phototoxic components removed or replaced. The products maintain exceptional support for cell function and viability during prolonged exposure of cells to blue light. Furthermore, BrightCell™ media products have very low levels of autofluorescence and photobleaching, dramatically improving the quality of data that can be obtained using fluorescent live cell imaging.

• MEMO^® is designed to replace DMEM prior to and during exposure of cells to blue light.
• NEUMO^® is a photostable medium which has been specifically developed for the culture of neuronal cells and can be used to replace Neurobasal^® media during live cell imaging experiments.
• SOS^® has been designed to directly replace phototoxic serum-free supplements such as B-27^®, NS21, SATO and N2 during live cell imaging.

Figure 1. Enhanced live cell fluorescent imaging of oligodendrocyte progenitor cells (OPCs) (A,B), cortical neurons (C) and hippocampal neurons (D) when cultured in BrightCell™ photostable media (MEMO/SOS, NEUMO/SOS) vs standard conditions (DMEM/SATO, Neurobasal/B27). Prolonged exposure to blue light leads to higher cell viability of NG2+ OPCs, cortical and hippocampal neurons when cultured in BrightCell™ photostable media compared with traditional media.

Figure 2. Fluorescent cell sorting of glial cells. Successful FACs sorting, culturing and differentiation of viable MBP+ GFP/RFP labelled oligodendrocyte progenitor cells (OPCs) from adult mouse brain is only achievable with NEUMO and SOS compared to Neurobasal^® and B27^®.

Figure 3. Fluorescent live cell imaging of neurons and astrocytes. 7-day old primary rat neuronal cells and astrocytes cultured in standard media (Neurobasal/B27) or BrightCell™ media (NEUMO/SOS) in dark or light conditions. Cells were stained with neuronal markers Tuj1, GFAP, and with DAPI after exposure to blue fluorescent light. Prolonged exposure to fluorescent light results in widespread cell death of both neurons and astrocytes with standard media whereas neuronal cells cultured in BrightCell™ media show no decrease in cell viability.

Figure 4. Live cell fluorescent imaging of neurons in BrightCell™ media (NEUMO/SOS). A) Primary rodent cortical neurons expressing mApple (EGFP) and a mutant gene Matrin3 (YFP) involved in familial amyotrophic lateral sclerosis, counter-stained with a nuclear dye (purple). B) Live primary rodent cortical neurons expressing mApple (red) and a mutated gene C9orf72 (yellow) involved in familial amyotrophic lateral sclerosis and frontotemporal dementia, stained with a nuclear dye (cyan). All neuronal cells grown in BrightCell™ NEUMO/SOS media.

Figure 5. BrightCell™ media preserves normal cell function of OPCs and human neural stem cells. A) Comparison of the effects of DMEM/SATO vs. MEMO/SATO media on OPC morphology and proliferation. B) Comparison of neural epithelial cell differentiation from human iPSC generated embryonic bodies using serum free bplements N2+B27 (top panels) or SOS (bottom panels) to generate neurons (β-III) and astrocytes (GFAP) after approximately 5 weeks of differentiation.

BrightCell™ is a trademark of Merck
MEMO^®, NEUMO^® and SOS^® are registered trademarks of Cell Guidance Systems LLC
Neurobasal^® and B-27^® are registered trademarks of Life Technologies

References

1. Stockley JH, Evans K, Matthey M, Volbracht K, Agathou S, Mukanowa J, Burrone J, Káradóttir RT. 2017. Surpassing light-induced cell damage in vitro with novel cell culture media. Sci Rep. 7(1): https://doi.org/10.1038/s41598-017-00829-x