Science Buddies: "Ask an Expert"

Hello, I am currently working on culturing breast cancer cells, I have not yet started because I don't have the materials but my teacher is getting them for me. I want to know how long I should freeze them for, to observe the results. I was thinking I should check them every two weeks for 1 week, or should I do it for a longer time? I will be making 3 plates for this experiment.

UPDATE 9/22/14
This is my procedure:
1. Sterilize hands and equipment with 70% alcohol, turn on UV light for 10 minutes to kill remaining bacteria in flow hood.
2. Incubate three flasks of breast cancer cells at 37 degrees Celsius and 5% CO2 level for 24 hours.
3. Place flasks in sterilized hood along with necessary materials and turn on pneumatic suction system to remove excess culture solution from culturing flask.
4. Rinse the inside of the flask using an automated pipette with dds or pps solution to remove any microscopic dirt and use pneumatic suction to remove excess DPS or PPS.
5. Add 1 to 8 ml of dissociation reagent (Triple E), only use enough to cover the bottom of the flask. let sit for 5 minutes.
6. Remove each flask from hood and tap each side of the flask while slowly shaking it side to side to loosen the cells that could’ve gotten stuck at the bottom of the flask.
7. Observe the cells under a microscope to make sure they are freely floating in solution. If they are deactivate dissociation medium by flushing the flask with F-12 culturing medium at room temperature. Drain off excess culturing and dissociation medium
8. Add 10 ml of culturing medium to the flask and place cells back into the incubator at 37 degrees and 5% CO2.

*That was the procedure that I came up with through research, I also saw a bunch of different procedures posted online and kind of changed it to fit my criteria. Also I decided I wouldn't freeze my cells anymore because I should look at them more than I said I would, but remember I would have to count them every time, I observed them which is the reason of why I don't want to do it every day, I would love some suggestions of observation times though. Also after doing this procedure several times, understanding it completely and knowing how to observe them, I would start part two which would be exposing my cells to anthocyanins to test if they slow down, prevent or cause an effect on cancer. I am currently trying to think of the procedure for the second part, because of the amounts of anthocyanins I would expose my cells to, Thank you.

Hello!

It is exciting to work with live cells! Could you perhaps tell us a little more about your study as well as your aims? It would help for us to offer advice.

A few questions:
1) Why are you freezing the cells to observe the results? If you are testing the cancer cell response to Anthocyanin, you would want to do that while they are actively multiplying.

2) Why do you plan to check on your cells so infrequently? Unless you are starting off with a very small concentration of cancer cells in your cultures, you will want do maintainenance on the cells for at least 2-3 days a week, depending on what you observe.

Hopefully, I can be more helpful when I know more about your project!

-crystalball

People often freeze cells in order to preserve them for future use. I am unsure as to why you want to freeze your cells, but usually, frozen cells are thawed quickly. You can learn about protocols for freezing cells from websites for companies such as life technologies and abcam . Although you can buy cell cultures from organizations such as the ATCC (www.atcc.org), the Coriell Institute for Medical research (ccr.coriell.org), these organizations restrict whom they sell their products to, so you might want to ask a professor or another laboratory scientist for guidance. Cell cultures from the ATCC and the Coriell Institute for Medical Research are less likely to be contaminated with other cell lines, bacteria, fungi, and yeast.

I know that they inhibit cancer cells and elevate their ROS levels killing the cancer cells but not harming healthy cells, also I am trying to figure out how much anthocyanins I should put on my cancer cell cultures once I have them, to test to see if they do slow them down or kill the cancer cells but not the healthy cells, I was thinking I could do 6 sample:
1. 20ul
2. 25ul
3. 30ul
4. 40ul
5. 45ul
6. 50ul

PLEASE! Keep ALL your posts in one thread so the experts can see all the questions and answers relating to your project without having to hunt through the whole forum list.

Thanks,

Sybee

Hi there,

I noticed your other post in another thread about anthocyanins and killing cancer cells. It is great that you are planning to treat the cancer wells with different concentrations of the compound, especially if it is not known what is the minimal concentration required to see killing activity. However, if you are going to have one individual plate of cancer cells to test each individual concentration of anthocyanin, I would make sure that I seed each plate with the same number of cells so that you can make comparisons between cell numbers before and after the treatments. Which, by the way, brings me to the question of what characteristics are you looking for in your treated cells? Are you going to count the cell numbers before and after treatment and see whether there is a decrease in the anthocyanin-treated cells compared to untreated cells? Are you going to treat healthy cells with the same concentrations of anthocyanin to see whether this compound kills healthy cells, or is only selective against cancer cells? You also mentioned something about anthocyanin elevating the ROS levels in cancer cells--are you planning on measuring ROS levels in untreated and treated healthy vs. cancer cells to see if there are any differences?

Since you are working with live cells, are you currently working in a university lab where you have access to all the needed equipment and resources? If you are, it would also be a good idea to talk to people in the lab who are helping you to get their opinions on your proposed experimental procedures!

Let us know if you need anymore help!

Connie

Hello,

I have a few questions and suggestions.

I saw your update on the procedure you are following to maintain the cells. Forgive me, but I am a bit confused about steps 7 and 8. Your cells will be growing indefinitely since they are cancer cells and you will want to decrease the concentration per plate/flask to make sure they do not get too crowded. Now, most people do this by splitting their cells---which involves detaching the cells as you have done in steps 5-6, centrifuging them down, taking away the supernatant, resuspending the pellet, and then diluting to the desired concentration in a new flask. Why are draining the cells in suspension in step 7? That will cause you to lose the cells you have been keeping. I would go on ATCC.org, as other experts have stated, to look up your cell line. They almost always have a protocol for cell maintenance attached. Thoroughly read it!

As for your actual experiment on Anthocyanin, I would absolutely do a dose response to start. This basically entails you using increasing/decreasing concentrations of Anthocyanin in different plates so that you can see a consistent and proportional change in some factor, maybe cell viability, throughout all of your flasks/plates. Unless there is prior literature that you have read where concentrations ideal for experimentation are given, I would use a large range so that you can get a sense of how your cells behave in the presence of the Anthocyanin. In addition, there are many assays available that allow you to quantify certain aspects of your cells, from cell viability to cell migration, etc. As the previous expert mentioned, if you are working in a university lab, do ask them for advice and thoughts on your procedures! If they have worked with the cells before, even better!

Let us know if you have any more questions!

-crystalball

That is a great idea to test a range of concentrations of anthocyanins. Have you read published studies to see what range of concentrations have been tested in other studies? It would be good to select your concentrations to cover the range that has been tested previously.

Have you decided how you will measure growth/inhibition of the cancer cells?

As the previous reply suggested, please put all of your questions and plans together in one post, then we will be able to help you more effectively.

Madeline B

How do anthocyanins inhibit cancer? I need to go to the cellular level but I can seem to find anything about this except for how anthocyanins elevate the ROS levels in cancerous cells and kills them

Hello there,

This review article may give you a better idea of why they think anthocyanins have anti-cancerous properties. Why don't you take a look at it and let us know if you have any questions or if you want to brainstorm ideas?

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2582525/

Connie

are there any other types of cells that are biosafety level 1, that I could purchase without a mentor instead of cancer cells? I would like the cells to be easy to handle since my teacher doesn't want anything hard because of our short period of time, also I would like the cells act in the same way a cancer cell would or maybe a type of maggot or insect?

Im not freezing my cells anymore. Sorry for the late reply.

Hello, we noticed you had two separate threads going for the same topic. We have merged the two threads into one. Please post all questions to only one thread, as that will help our experts answer your questions based on what has already been discussed. It looks like you are off to a great start to your project, and you have received a lot of excellent advice. Good luck, and thanks for using Science Buddies!