Protocol Online logo
Top : Forum Archives: : Cell Biology


Quick Questions:
Does a HEPES-only solution or a HEPES-containing solution, such as artificial cerebral spinal fluid (aCSF), that's been variously exposed to light pose a risk to cells IN VIVO (i.e., no exposure to light once the aCSF has been introduced to the extracellular environment)? Would it be considered important to prepare, store, and administer these solutions with minimal light exposure if the target cells are never exposed to light?

More details related to questions:
I was hoping someone could "shed some light" on the photo-/cyto-toxic activity of HEPES. We've been using it for years but only recently came across references citing its potentially toxic effects. In our lab, HEPES is a component (10 mM) in aCSF (recipe appended below), which we use as a vehicle for drug delivery via intra-cranial micro-injections. One or more of the neuro-active agents that we dissolve in aCSF are light sensitive and we do prepare these drugs in amber vials under low light conditions. When not being used, we do store our aCSF in a (dark) refrigerator, though any given batch might get drawn from on a daily basis (i.e., light exposed 15-30 min per day) for several months before a new batch is made.

I did a quick read of a 1985 Zigler et al. paper, "ANALYSIS OF THE CYTOTOXIC EFFECTS OF LIGHT-EXPOSED HEPES-CONTAINING CULTURE MEDIUM." It concluded that 1) exogenous H202 added to non-irradiated medium is less damaging than is irradiated medium with the same H202 concentrations, and 2) irradiated HEPES-containing media had a stronger inhibitory effect than did cultures in which only HEPES (10X) and riboflavin (l0X) were irradiated. We don't work with cell/tissue cultures, but we do inject variously irradiated (light-exposed) HEPES-containing solutions in vivo, that is, into an extracellular environment (a non-irradiated medium) bathed in many of the micronutrients found in benchtop culture media.

Some noteworthy differences between our work & cell cultures include: 1) in vivo cells are never irradiated though our HEPES-containing aCSF is routinely so; 2) we work with lower HEPES concentrations (only 10 mM vs. 25 mM); and 3) we inject very low volumes of a HEPES-containing solution (0.25 - >2 microliters) in vivo wherein the neuro-active molecules bind to target receptors or ion channels while the vehicle becomes diluted (or washed away) across the extracellullar milieu. Our animal subjects could conceivably receive one such injection up to 5 times per week for a month or longer. Please note that it's not uncommon for the "quality" of the injection site to deteriorate (gradually or quickly) over time until drug effects are lost. Conventionally, this "effect" is attributed to gliotic scarring of the injection site caused by the repeated insertion of the needle into live tissue. But now I'm a bit curious if some of this local tissue damage could result from low-level toxicity introduced in our aCSF. I should note here too that treatment & control injections follow identical protocols, differing only in presence of drug; that is, we also run control experiments (aCSF-only) that routinely show that our results can be attributed to the drug's effect, not its vehicle.

If anyone knows of a more appropriate venue (listserv) to post this question please let me know.

Thanks for any helpful comments.


Lepe-Zuniga et al. reported a phototoxicity of HEPES when exposed to ambient light by the production of hydrogen peroxide. For best repeatability of results it is then strongly advised to keep any HEPES containing solution in darkness as much as possible.

see theses refs :
* Toxicity of light-exposed Hepes media. Lepe-Zuniga JL, Zigler JS Jr, Gery I. Journal of Immunological Methods. 1987 Oct 23;103(1):145.
* Analysis of the cytotoxic effects of light-exposed HEPES-containing culture medium.
* Zigler JS Jr, Lepe-Zuniga JL, Vistica B, Gery I., In Vitro Cell Dev Biol. 1985 May;21(5):282-7.

from wikipedia webpage