Apoptosis

CQ

Hi can anyone help me, i;'m trying to induce apoptosis in three cell lines using cycloheximide, but have been unsuccessful so far....well i'm not getting any DNA laddering. I have used a 100ug/ml of cyclo for 48 hours and still they wont die....... does anyone know any other methods of apoptosis induction with reagents that are quite cheap as my budget is small!!the cell lines are endothelial cell, synovial fibroblasts & chondrocytes

2Scoops

H2O2 should do it and is cheap.

CQ

Thanks, do you have an idea what conc or volume i should use in 24 well plates, that hold about 1ml

2Scoops

Depends what you're looking at - it's always wise to do a dose-response curve and see what comes out best. If you're only after a very basic induction of apoptosis, I'm certain you'll find other papers using ECs, fibroblasts etc. which will give you a ballpark figure.

CQ

Thank you, will have a look
For now i just want to be able to induce apoptosis, then i will transfect the cells with my protein & see if this influences the results

liberal

Interesting, some kind of super protein that makes the cell invicible?

CQ

well thats what i'm trying to find out, if it does have any effect, but first i need to make sure i can induce apoptosis first. Then i can start the experiments!!

liberal

Have you studied oncology?

CQ

no, not studying oncology, doing research into rheumatoid arthritis

Staph

What about just serum starving them, removing all serum from their media. It has worked for me previously in rat endothelial cell.

CQ

That was my next course of action while i still have cycloheximide available, do you think 48 hrs serum starving is too long would 24 hrs be enough

cgc5483

CQ wrote: »

That was my next course of action while i still have cycloheximide available, do you think 48 hrs serum starving is too long would 24 hrs be enough

I would suggest that there is something wrong with the cycloheximide you are using. 10 ug/ml should be sufficient to induce a significant level of cell death in most cell lines at 24 h so am guessing that if you are actually using 100 ug/ml then something if up with the cycloheximide or else you have the calculations wrong

Won't use H2O2 as it mostly induces necrosis and only induces apoptosis in a very narrow concentration range and can vary from day to day.

Serum withdrawal as previous poster said should be quick and cheap. 24 h should be sufficient especially if you want to look at cytoprotective effects as you don't want a complete massacre on your hands.

Another quick and cheap drug you may have would be actinomycin D.

By the way you need to be careful interpreting your results as if your protein doesn't affect the levels of apoptosis it doesn't mean it's not anti-apoptotic. You really need to try several stimuli that activate apoptosis in a variety of manners.

Good luck!

Edit: Just realised that you are monitoring for apoptosis using DNA laddering. In my opinion it's not the best way as technically it's quite difficult to do especially in some cell lines; not quantitative either so difficult to measre the effects of your protein. If you want something cheap and have access to a fluorescent microscope then you could stain the cells in the well with Hoechst and look for condensened and fragmented nuclei indicative of apoptosis.

liberal

From where I'm sitting you're talking about necrosis......

derek27

Your appraoch is not inducing apoptosis. Cycloheximide can induce it, but has wide ranging effects that may interfere with your particular endpoint assessment. Look at the link below for a range of apoptosis inducing agents including information on the other effects that the chemicals/agents are having. I'd recommend Etoposide for the purpose of studying rheumatoid athritis, and it's cheap so won't eat into your budget.

http://www.biovision.com/updated/apop_inducers.html?gclid=CP2HnLn_9J0CFU0B4wodDDjIIw

hugoline

cgc5483 wrote: »

Edit: Just realised that you are monitoring for apoptosis using DNA laddering. In my opinion it's not the best way as technically it's quite difficult to do especially in some cell lines; not quantitative either so difficult to measre the effects of your protein. If you want something cheap and have access to a fluorescent microscope then you could stain the cells in the well with Hoechst and look for condensened and fragmented nuclei indicative of apoptosis.

I would 100% agree with cgc5483, DNA laddering is not done anymore these days (and neither are MTT or LDH assays), although easy to perform, they are usualy not accurate at all.

Have a read of this article, for other methods of detecting apoptosis (and NOT necrosis), authored by the leaders in apoptosis research.

If you have a good light microscope you can even 'quantify' apoptosis according to the phenotype (cell shrinkage, detachement, blebbing, nuclear condensation) depending on the cell line some of these features are harder or impossible to see (and sorry, I haven't worked with the cell lines you are using so wouldn't know)

Drugwise: I am not surprised CHX doesn't induce apoptosis, it inhibits protein translation and only works on a few cell-lines by itself. (it is very potent with TNF which is expensive though).
Besides the already mentioned drugs, UV is another cheap and effective way of inducing apoptosis. Just use the box where you detect the DNA gels and do some test as how long to leave the cells (still in their dish) exposed to UV (usualy 30 sec to max 5 minutes) to see apoptosis within 8-12 hours. Again, too long and it's necrosis.

CQ

Thank you for all the help, i definitely think that apoptosis is induced and i'm just not getting the DNA ladder, just the HMW DNA is showing up even though i'm using a sucide track assay to isolate the fragments!

I do have TNF-a available, but i will be using that to induce an inflammatory response in my cells after transfection

Staph

CQ wrote: »

That was my next course of action while i still have cycloheximide available, do you think 48 hrs serum starving is too long would 24 hrs be enough

I starved my cells between 3 and 5 days. Best of luck!

Kevster

Apparently, inducing mitochondrial fission can induce apoptosis too.

Tree

Kevster wrote: »

Apparently, inducing mitochondrial fission can induce apoptosis too.

Cell full of calcuim \o/ go my pretties, interact with the big rosettey protein complex thing (guess who forgot the last segment of her cell signalling module of last year)

Kevster

....odd, I'm just looking to get into a project which involves mitochondrial fission/fusion. I assume that you're referring to GTPases here, and that the 'big rosettey protein complex thing' is a mitochondrion? Anyway, calcium does indeed propagate some signals, intracellularly.

CQ

I think i've manage to induce the apoptosis now, and hope to get a ladder, if not then i will have to try some other way of detecting apoptosis which isnt too expensive. Judging by observations with a light microscope apoptosis is induced.

Tree

Nay, not ye old mitochondria, but the apoptosome (caspase 9 complex).

My memory failed the other night

charlietangodel

Could you use ceramide, DMS or Heat?