Science Buddies: "Ask an Expert"

Hello,
I am doing a science project about alzheimer's disease. it will involve culturing macrophages and stimulating them with amyloid beta, then adding neurons to the culture to create a mixed culture. basically i want to find out how the cytokines the macrophages release (hopefully) will affect the neurons [e.g. apoptosis occurs]. so, basically, are there any techniques for counting one cell type specifically in a mixed cell culture? like doing something prior to combining the cultures to make the neurons to appear different (some kind of stain?) under a microscope so they can be counted apart from the macrophages?

thanks!

anna

Hi Anna,

Wow, this sounds like a very interesting and complex project.

You are correct, there are markers that are specific to neurons that can be stained as well as those that are specific to macrophages that can also be stained. Often its done using a fluorescent tag that is bound to an antibody that binds to the receptor. Then you use a special microscope that can show the different fluorescence. (http://serc.carleton.edu/microbelife/re ... romic.html). You will need to be careful though that the stain doesn't effect the phenomenon you are trying to study - apoptosis. Studying apoptosis also involves using stains to see what is happening to the cells.

If you are working with mammalian cell cultures I assume you must have a lab mentor. This would be the best person to discuss the details of your options for staining with.

Best of luck,
Caroline

Hi Anna,

That does sound like a great project. I just have a few things to add on top of the previous response, which is excellent.

1. Here is one paper to get you started on your search for the right staining technique: http://www.ncbi.nlm.nih.gov/pmc/article ... ool=pubmed. There are, of course, several methods of staining since there are several usable antigen targets. So, look for articles involving methods of macrophage staining, and see which one seems to fit your project the best, given the materials that you are using.

2. One thing that you will have to make sure of (and talk to your mentor about this, he/she will have a much better idea about this than I do), is that the staining does not interfere with the normal functioning of the cell if you want to stain and trace it in vivo. Often times, staining a cell can cause it to do funny things or rigidify, etc. Look through the papers to see how different staining techniques might affect your specific macrophages.

Hope this helps!
Aaron Lin

Hey there, Anna!

Unfortunately, I do not have the answer to your question. Although, I would love to notify you that this is a very creative and interesting project!

I just had some questions for you, since I'm having sciencefairideagenerating-block

1. How did you come up with this idea? (Even if it's a long story, I'd love to know!)
2. Do you have a mentor? If so, are you using a lab for your experiment?
3. Are you using specialized products/equipment? If so, how did you obtain them?

Thank you very much, and good luck!

Haha, thank you!
1) To be honest, I don't really recall exactly how I came up with my topic. I just remember many, many articles, textbooks, magazines...lol.

2) I have a supervisor who will oversee everything I do, however she did not mentor me in the sense of brainstorming or helping with protocols. I am using several labs actually - i need access to several pieces of equipment, so yeah.

3) The main obstacle in my project was $$. Thus, I had to get donations for everything. It was difficult and time consuming, but very necessary as my parents aren't rich. Equipment = nearby university.

If you're not quite sure where to start, Scientific American is great b/c it's pretty understandable as well as interesting. But then again, it's fairly mainstream and unlikely to give you specialized information so you should just use it as a starting point for brainstorming. Maybe you are already interested in a certain field - if so, use google scholar to find interesting articles about your topic that may lead you to a project idea. If you keep doing research, eventually you'll get to the point where you can create an original project idea from what you have learned.

Good luck - contact me again if you need help!

edit - also, contact professors at local universities! it didn't work out for me, but it could for you. if you have connections to universities, definitely use them.

Ooh, thank you so much!

Yeah, money is a big obstacle. I'm just in middle school right now, although I'm looking towards a more advanced and original project. Check out my topic, perhaps? https://www.sciencebuddies.org/science- ... =25&t=5967

Yes, I'm in the same situation as you - reading, researching, reading, researching. I've done a lot of research, but I'm getting overwhelmed - I've been researching for a month or two now :S . AHH! Haha. Did you have other possible project ideas? How did you narrow it down?

So my last research resource will be the Scientific American (I've only entered the homepage on my Internet but already it's looking amazing!). As for Google Scholar, when I first used it, I thought it was just a bunch of scientific term. Now, I think I know how to actually use it for project brainstorming... although some of the papers look complex. Having project limitations and restrictions (check out my topic ), it's slightly hard to find the perfect project. You're lucky - you have a nearby university! I live in a small town, but my dad is an engineer and we have a community college. Hey, and don't underestimate 'small town'. My district science fair is insanely competitive (well.. not insanely, maybe really). Haha.

Last question - how much time in total did it take you from the start of your science project to the end? How much time before the fair did you start? How much time did it take you to do project research, background research, the experiment, etc etc. Sorry for all the questions and for being nosy!

Thank you SO SO SO much!

cool, i happen to be in middle school also.
once you've done some general research on a few interesting topics, you should consider going into depth on one of them. read journal articles ,etc.
just keep reading the papers and look up terms you don't know. it'll get much easier, trust me.
yeah, i do live in a good location for research - transportation is still a hassle though since both my parents work.

well...i'm still working on my project! i'm just beginning with the actual lab work, although i've been resarching, fundraising, and planning for about 6 months.
i started trying to find a topic for this year right after the state science fair (cssf) ended, around may. i only finalized this project around july though.
my experiment will take me roughly 3-4 weeks in total, though data analysis will take me about 2 weeks probably.

when is your project due?

oh, btw, i was reading your other thread - microbial fuel cells sound very interesting. two girls at my regional fail did a project about that and they got 2nd or 3rd at isef.

Wow, cool! What grade are you in middle school? I'm in grade 7. Oh man, your project sounded so amazing that I thought you were older! Does your middle school go past grade 8, is that why you posted this in the 9-12 section?

Ah, I see. You put a lot of work into this! Did you do the fair last year? What was your project last year? Thanks for answering my questions

Arrrgh, this year I'm actually very disappointed in myself. I did pretty well last year and was going to start my project early this year, although I delayed it and started around December. I've been researching for a month now. Mine's due April 2nd. I'm planning on finalizing my idea by Saturday, then getting materials and equipment+doing background research by the first weekend in Feb. I will conduct my experiment until the third week of Feb. For the last two weeks, I will analyze data and make conclusions. Lastly, I'll concentrate on the time remaining to practice, make the display board, etc etc. When is your project due?

ISEF?! WOW! Do you remember exactly know what they did?

I'm in 8th. I thought I would get better feedback if I posted in 9-12. Thanks for the compliments
Yeah, I did do science fair last yr. I can't tell you the specifics b/c I don't want to identify myself (all the cssf projects are online ), but it was a behavioral project.
Hey, you have until April 2nd! That's a lot of time - mine is due middle of march. You have a good plan - good luck with that.
I don't remember exactly what their project was - however, you can look them up on the cssf or isef sites by searching microbial fuel cell.
So, what exactly are you doing with microbial fuel cells? They sound really cool.

Haha, no probs! That sounds pretty cool!
Yeah, I guess you're right. Okay, so I searched the ISEF and CSSF websites and found a few links. Turns out that some winners did use the MFC (microbial fuel cell)!
Well, I'm not completely sure what I'm going to be doing with them yet. I'm either doing that or basing my project on solar cell output. I'll give you more info on my topic as I move along.
How about you? Did you have any alternate projects? What was your inspiration article for this project of yours? Tell me how you did your fundraising.
So what are your educational plans? Are you looking towards ISEF(ISEF is grades 9-12, right?)? Do you have any educational goals such as a university you want to attend or a career path?

yeah, i did have a few less complex project ideas that did not need university lab equipment/expensive materials, but i wasn't as interested in them - i really wnted to do this project specifically.
there wasn't an inspiration article really - it was kind of a compilation of ideas i had as well as current research going on in this field.
fundraising - simple enough. email a bunch of companies and ask for free stuff.
isef would be great, but who knows? i'll just try to do the best i can.
haha, i'm in 8th grade. i have no idea where i want to go to college...i'm just trying to get into a good high school! being a scientist would be cool, though.
what about you?

Hey!

So I see. I totally understand where you're coming from - there were a few projects I wanted to do but couldn't since they were expensive, time-consuming but I was REALLY interested in them. Unfortunately for me, I started out considerably late, so I could not pursue those experiments. Lucky for you, you started early and got access to resources!

Ahh, I see. So which companies did you email? What kind of 'free stuff' did you want, or which stuff did they give you?

Haha, yeah it sure would! Me? Well my family is big on education, although my hobbies aren't limited. I'm attempting to pursue my parent's/my dream of attending a good university, and so I'm thinking of studying law/dentistry. Idk, i'm still young, but, well, I can dream!

i don't remember exactly which companies...a lot. just the materials i needed, such as assay kits, arrays, buffers, culture medium, cells, peptides, etc.

you still have a ton of time - it's due in april. you can still contact companies regarding donations, etc.

cool, what college do u want to go to?

Oh, that's cool! So how did you convince them to donate? Did all of them donate?

An advantage for me is that my dad can bring back some supplies from his engineering plant, so for example last year I used a conductivity meter from his work. I finalized the MFC project (well, my dad's going to read my ideas over and just double check. Unless there's a major obstacle I'm going to do an alternate project.) So I bet he can access some of the equipment, and I can use a multimeter this year.

Well, I'm not exactly sure which one. There are a lot of good ones, but most are far away from home. Specific college research will be done in grade 11 or 12 haha.

Just wanted to comment on how great you both are doing. It's very encouraging to see middle school students so interested in science and attempting pretty complex projects on cool topics. Try not to worry too much about not being able to do the type of project you ideally want to do. Even established scientists often can't do the experiments that they would like to do in an ideal world because they wouldn't be ethical or would cost too much so they have to use model systems.
Some of the best experiments can be very simple but just a new way of looking at things. And doing a good job with a project that only needs simple equipment can show a prospective mentor how you have applied the scientific method. Or help you get a place on a science camp or summer program at a university.
Just keep that interest alive. Its great that you have started finding scientific articles about your area of interest. Another place you might find information is scientific societies. Some of the societies have junior membership that lets you read articles from their publications and attend meetings and meet scientists working in that field.

Best of luck with your projects,
Caroline

Hi Ms. Thorn,

About my cell counting, I was wondering - would you know if FACS (fluorescence activated cell sorting) would be applicable to this situation? The description sounds like it is, however not having worked with it before I'm not sure. It seems ideal b/c it is very fast as well as being quantiative.

Thank you so much!

I just find doing a science fair project so intriguing that talking about experiences and ideas are wonderful! Thanks for the advice and compliments!

Anna, read my last post, the one before this. I think you missed replying to it haha.

Hi Anna,

I don't know a great deal about FACS but you would have to have your cells in solution to do it. From what you mentioned in your first post it sounds like your cells will be growing attached to a plate so you would have to remove them with an enzyme first. You would want to make sure that the cells counted are all alive at the end so it doesn't obscure those that underwent apoptosis. It may sound faster but troubleshooting how to get the FACS to work with your system might be tough and end up taking a lot longer than a more visual method. There are a variety of computerized methods now for counting from captured images. Just my thoughts, maybe another expert who does more cell bio can help?

-Caroline

Hi Anna,

Yes, FACS would definitely be one possibility, though you would have to work out some of the more technical details, as Caroline points out.

FACS isn't especially different from many of the staining methods previously described. Basically, you want to identify a few (usually at least 2, the more, the better) cell surface markers (or a combination of markers) that are specific to the type of cell that you want to sort out. For example, one of the more recent combination of markers for hematopoietic stem cells (HSCs) is CD150+, CD41-, CD48-, meaning that cells with high concentrations of CD150, low conc of CD41, and low conc of CD48 are HSCs (this combination is pretty unique to them). I have no idea what a potential combination for staining neuronal cell surface markers would be, but I'd imagine you could find it in the staining literature given previously. This paper (http://www.ncbi.nlm.nih.gov/pmc/article ... ool=pubmed) posted earlier seems like a pretty good start.

Note: there will not be just a single combination that works perfectly. Almost no combination is perfect, and there may be several that are pretty good. If you look at the history of the cell surface markers for HSCs, it's pretty jumbled but improved significantly. In the distant past, they just sorted out cells with any mature cell surface markers and found a few HSCs per billion or so of cells. More recently, one code has it at c-kit+, Lin-, Sca-1+, which results in 20% HSCs, and the previous method that I described with CD150+, CD41-, CD48- gives roughly 50% HSCs. So definitely keep searching until you find the best combination for your project.

After you pick your cell surface markers, you need to stain the cells with fluorescently-tagged antibodies against those surface markers. Stick your sample in the FACS machine, and it flows the sample through a few chambers, where a laser excites the tags, which in turn emits photons. The photons are detected via photomultiplier, and that allows the machine to sort each different color tag into a different chamber, allowing you to sort out cells by cell surface markers. The computer readout then tells you how many cells had which types of markers, and you can get the sorted tube that you want out of the machine at the end, very much alive and happy.

With respect to the technical details, I have never actually done FACS myself, so I can't give you a perfect picture of how well your cells will be. As Caroline points out, you do need to trypsinize (or some other method; the gentler, the better in your case if you want to keep them alive) the cells to remove them from the plate, but that shouldn't be too bad. The antibody staining won't please the cells, but I don't believe that it would kill most of them, but again, try to minimize damage to the cells to prevent confounding apoptosis. So, there are definitely some technical hurdles with FACS, but some of them are from the staining, and the nice thing about FACS is that you get your sorted cells back at the end, which allows you to do more cool stuff with them. Again, fantastic project idea!

Hope this helps!
Aaron Lin