I am planning to treat HT-22 cell line (neuronal) with NMDA. How do you determine the cell density and NMDA conc. in order to achieve 40%-50 cell death and what precautions must be taken while performing the experiment as well as while analysing data. Can anyone share his/her protocol for dose standardisation studies.
I assume that you want to determine a concentration/effect curve (often inaccurately referred to as a "dose/response" curve - inaccurate because you generally do not give drugs as a total amount (dose), but rather at a given concentration). The first thing to do is precisely define your "effect", i.e., it could be number of dead cells per unit area at a prespecified time post application of NMDA, or concentration of some marker of cell death at a pre-specified time, etc. There are many ways to measure or estimate cell death/survival - there is no doubt something for which you (or a neighboring lab) already have the necessary equipment.
Some things to keep in mind:
1. keep your glycine concentration consistent (I recommend aiming for physiological concentrations, but if you want to maximise sensitivity to NMDA, may want to go higher). 2. Think carefully about what you want Ca++ concentration to be - this is a critical player in excitotoxicity, and something that is generally not appreciated is that extrcellular concentration of Ca++ falls dramatically temporarily in areas of insult as it enters stressed cells and leaves the extracellular space (contrast this with how much iin vitro work is done, where extracellular Ca++ is essentially and stays high because of the relatively large volume of solution bathing the cells).
3. Temperature - another significant factor in whether a given insult will cause irreversible damage or not.
4. Your glassware/plasticware - many plastics leach out pharmacologically active substances. I don't know if any NMDA antagonsts have been identified, but you'd want to be consistent with whatever may be affecting your prep.
I've not carried out in vitro experiments for some time, so there are no doubt other important details.
I hope this is helpful
Thank you eric this is very helpful for a beginner like me and i really appreciate the detailed explanation given by you. I now have a few more queries.
1) to induce excitotoxic neuronal death , i would be exposing HT-22 cells with NMDA dissolved in growth medium (DMEM by gibco) which inherently contains 1.8 mM Cacl2. I would like to know whether this Cacl2 dissociates to generate an equivalent amount of Ca++ ions ie 1.8 mM and is this concentration sufficient to induce neuronal death. ? or will i have to expose neurons to NMDA dissolved in HBSS.
2) i would be performing an MTT assay for determining the effect of various NMDA concentrations on cell viability. I am planning to seed cells at various concentrations ranging from 1000 - 10,000 cells/ well and treat them with multiple NMDA concentrations from 100 nM - 1uM for 6 hrs. Is this experimental design correct or am i missing any critical thing.
3) Can i use the same experimental set up for determining the effect of Kainic acid.
Shashank:
I am delighted that my comments were helpful. I must admit that I have not worked with HT-22 cells, or actually any cultured cells - just acutely prepared hippocampal slices (rat and guinea pig). I know there will be some differences (e.g., slices are less sensitive to bath-applied glutamate because of presence of glia and transporters "protecting" glutamate receptors), but in terms of sensitivity to NMDA and Kainate I'd expect my experience to be relevant:
in our experiments,we acutely made slices ischemic/hypoxic (solutions saturated with N2 instead of O2, and glucose dropped from 10mM to 0.5 mM) until they exhibited a massive rapid depolarization. Slices maintained in 1mM Ca++ did not recover from this depolarization (at least not within an hour). Note that we were also doing our experiments at 36.5 degrees C. Slices were made ischemic/hypoxic in the presence of 0.2mM Ca++, then returned to 1mM Ca++ when oxygen and glucose were reintroduced, all recovered to about 60% within an hour.
So:
1) I don;t know the solubility of CaCl2 in that buffer, but I'd expect 1.8mM to be sufficient (It certainly is physiologically relevant anyway)
2) I've not used the MTT assay, but, I think an important thing for you to decide upon is how long to wait before assaying (i.e., do you want to look at acute cell death or a delayed cell death?). I assume your work is related to stroke, and if so In vivo, one gets both types of cell death with stroke (e.g., the core vs. the penumbra), and I think the delayed cell death (the penumbra) would be the target of any treatment after stroke.
3) If your cells express KA-sensitive receptors, I would guess that you could make kainic acid work (wouldn't necessarily work with the same concentration as used for NMDA, and might show different Ca++ sensitivity).
Good luck, and I hoep you get some input from people who have worked with HT-22 cells.
Eric
Hi eric thanks again , your in depth analysis is just amazing . Is it possible that excitotoxin's when applied to neurons can be endocytosed causing additional toxicity apart from Ca2+ ions entering the neuron through receptors.
Shashank: Thank you for your kind words.
The idea of an intracellular excitotocix target is a very interesting one - I am not aware of any evidence of that related to Excitotory Amino Acid receptors, but that may reflect my ignorance of the latest in the field, or this may be something waiting to be discovered!
As for toxicity other than via directly causing Ca++ entry, there are metabolic effects via metabotropic receptors, and I think it is well established that activity at the traditional ionotropic receptors can have metabolic effects independent of ion flux (I've not followed that topic and so can't say any more about that).
Something that is little off-topic from your question, but might be of interest, is that it has been demonstrated that quisqualate can be taken up and re-released via heteroexchange, presumably involving glial cells. I authored 2 articles about this and first proposed that mechanism. If you are interested, do the following search in PubMed: "((harris e) OR (koerner j)) AND quisqual".
In any case, good luck!
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