Abstract
Light is extensively used to study cells in real time (live cell imaging), separate cells using fluorescence activated cell sorting (FACS) and control cellular functions with light sensitive proteins (Optogenetics). However, photo-sensitive molecules inside cells and in standard cell culture media generate toxic by-products that interfere with cellular functions and cell viability when exposed to light. Here we show that primary cells from the rat central nervous system respond differently to photo-toxicity, in that astrocytes and microglia undergo morphological changes, while in developing neurons and oligodendrocyte progenitor cells (OPCs) it induces cellular death. To prevent photo-toxicity and to allow for long-term photo-stimulation without causing cellular damage, we formulated new photo-inert media called MEMO and NEUMO, and an antioxidant rich and serum free supplement called SOS. These new media reduced the detrimental effects caused by light and allowed cells to endure up to twenty times more light exposure without adverse effects, thus bypassing the optical constraints previously limiting experiments.
Highlights
For 40 years, ambient light has been known to be toxic for cells in vitro[1] and yet recent advances in new methodologies utilizing hazardous levels of light for non-invasive control of cells have rapidly evolved, such as optogenetics[2], super-resolution imaging[3, 4], ion and voltage sensitive imaging[5], live cell imaging[4, 6,7,8,9] and light triggered drug delivery[10]
To address the effects of light on non-transfected cells, we developed a customized plate housing 6 light emitting diodes (LEDs) emitting blue light (470 nm) that sits on top of a standard 6 well culture dish with a controllable output power (W: 0.1–1.5 mW/ mm[2] at cell surface), flash duration (τ: 1–10 ms) and frequency (f: 0.1–90 Hz), operated from a power unit housed outside the incubator (Fig. 1a)
Optogenetics is intensively utilized in neuroscience, so we began by irradiating primary rat cortical neurons with flashes of light at intensities typically used to activate ChR2 transfected neurons
Summary
For 40 years, ambient light has been known to be toxic for cells in vitro[1] and yet recent advances in new methodologies utilizing hazardous levels of light for non-invasive control of cells have rapidly evolved, such as optogenetics[2], super-resolution imaging[3, 4], ion and voltage sensitive imaging[5], live cell imaging[4, 6,7,8,9] and light triggered drug delivery[10]. Optogenetics is a branch of synthetic biology involving the genetic introduction of light sensitive actuators to control neurotransmission[13], subcellular signalling cascades[14, 15], and gene regulation with temporal and spatial resolution[16,17,18]. Common optogenetic actuators such as channelrhodopsin-2 (ChR2)[13], melanopsin (OPN4)[19], cryptochrome-2 (Cry2)[15], and light-oxygen sensitive proteins (LOV)[15] rely on blue light for their photo-activation. To fully utilize the advantages of these novel methods, we have focused on finding efficient solutions to blue light (470 nm) induced toxicity; by utilizing neural cells due to their sensitivity to light and the exponential increase in the usage of light stimulations in neuroscience
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