I want to make a growth curve between control and test PC9 cell, in vitro. So please suggest me the method for it.
Thank You in advance.
The easiest way is to do an "MTT" assay.
SUPPLIES NEEDED:
Microplate reader
Media
Cells
Pipette and preferably multipipette: e.g. 100 ul
Small pipette for adding drugs if needed. Should be in small enough volume so that it does not affect the assay.
For instance, use a 99 well micro plate. Get MTT/tetrazolium. Everyone has his or her own way to do this, but this is how I did it:
First, you need to figure out the concentration of cells that will allow your cells to grow for at least a week without overgrowing. The way to do this is to put media in each well of your plate except for your left column. Perhaps put in 100 ul (microliters) in each well. (You need a pipet, better yet, a multi-well pipet of proper size.
Then put, for instance, 200 microliter of cells in the left row. The cells should be in the same media as the other wells, of course.
Make serial dilutions of the cells: Use a 100 ul pipet. Take 100 ul from the 1st column. This will leave 100 ul in these wells. Then, pipet this 100 ul of cells into the next column and mix the cells and media gently. You now have a 1:2 dilution. After mixing the cells, take 100 ul from the 2nd column and add it to the third column. The 3rd column will now have a 1:4 dilution as compared to the first column. Continue until you have made up dilutions for all the columns.
. Allow the cells to grow up for a few days and test 2 or 3 days for each row. I would recommend testing 3 rows using the MTT assay after 1, 2, 3.... 7 days to identify the relationship between number of cells plated and cell growth. You can put the MTT in a few rows and then measure on a microplate reader without affecting the other cells.
When you test a drug or other condition, you want the cells to be in the log phase of growth. By doing the dilutions of cells, you will find out what starting concentration is needed so that your cells are in log phase growth on the test day. If they are in log phase for 5 to 7 days, you will probably be fine as most drugs seem to show effects within this time frame.
MTT ASSAY: Use the manufacturer's guide for the MTT assay. Add MTT to each test well and incubate. Then check on a microplate reader. (see notes below and at Wikipedia Link to MTT assay.)
If you don't have a microplate reader, they might be able to buy one used from a used equipment dealer. You can also count cells under a microscope if you are a crazy or have a lot of free help.
A lot of researchers seem to randomly choose the number of days to grow cells. This has led to errors in some articles, in my opinion. If everyone is testing cells at five days, better do it that day even if you get the wrong results, then compare your accurate results, but be diplomatic if you are disagreeing with famous scientists. These preliminary studies will also give you familiarity with your cells and pipettors.
2nd: After a little study and aggravation, you will figure out the best media, cell count, and assay day for your cells. Each cell line is different. To find the best media, look at articles or else the American Type tissue Culture website or the website of other places that sell cells (Poetry here?).
3rd: test your drug or whatever. Put 100 ul of cells at the appropriate concentration in all of your wells but put 200 ul in the first column. Put your drug at a high concentration in the left column. Then making serial dilutions in the same way. Since all the wells have the same concentration of cells, this will dilute only the drug. Of note, make sure that all your wells do have the same concentration of cells by gently swirling the cell source before each time that you add cells. Also, obviously, make sure that you have a control column without drug but WITH the drug solvent.
I testing doing these assays without adding MTT and got about the same results, though it was hard to determine the lower levels of cells.
4th: determine the IC50 for drugs. This is the "inhibitory concentration" or concentration that inhibits cell growth by 50%. Other assays can be used. The usual comparison is IC50, but IC10, IC90, etc. can be interesting. You determine the IC50 by using the untreated column as the control. If there is an effect from the solvent for the drug, you will notice this also.
In analyzing your results, be sure to do at least three rows and determine the mean and standard deviation of your results. Repeat at least 2-3 times on separate days. If your results are markedly different, repeat until you are sure that you are getting accurate results. Take pictures of the microplates as they are pretty and might help you get published. I have some pretty pictures I should probably get around to publishing.
Also, make sure that you have considered how you are adding your drug or other additive. Make sure the drug is in solution and consider whether your solvent (e.g. alcohol or DMSO) could be affecting your system.
MTT NOTES: You might need to test the proper concentration for the MTT first by doing dilutions of cells. You want the MTT to be on the slope of the curve at the proper concentration. You can determine this while you are doing the other control experiments. Put cells at different concentrations into the microplate and use the MTT to find out what combination of MTT and time of assay is needed to make sure that you are on the "slope" of the assay. You need to be able to distinguish different concentrations of cells of course. Also, some cell lines are metabolically different and need different conditions.
It will take a few times to repeat this..
Try PrestoBlue assay as recommended by the suplier. It is faster and better than MTT.
To determine the cell growth there are a number of assays
1. MTT/WST-1 assay
2. ATP quantification assay
3.Trypan blue assay
4. AO-PI staining
5. AO-EtBr staining
6. Hoechst staining
etc
But MTT assy is the easiest method.
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