Hi everyone. I am gonna use an Acid Sphingomyelinase inhibitor to inhibit ASM and see the its effects on endocytosis of a receptor. I am using 1-Aminodecylidene bis-phosphonic acid that is selective for acid sphingomyelinase over neutral sphingomyelinase (IC50 = >100 μM ).
I can not understand what is IC50>100uM meaning. I used 15uM final concentration but I did not see a big difference between samples with and without ASM inhibitors.
I am wondering if I need to increase the concentration?
Thanks
Hi Akram, I don't know anything about the specific enzyme or inhibitor you are using but in general, IC50 marks inhibitor concentration that inhibits 50% of enzyme activity. In your case it seems that you need to increase your inhibitor concentration at least up to 100 uM. It would be a good idea to probe different inhibitor concetration (using 100 uM as the lower one) and test enzyme activity to determine optimal inhibitor concentration.
Hope it helps
Angel Gaudioso
IC50 > 100 um means that the inhibitor concentration needed to obtain 50% inhibition is more than 100 micromolar. This is a very poor inhibitor and you will have to use much higher concentrations to get efficient inhibition of ASM. Can you purchase a better inhibitor?
Your dose/concentration is less (15uM) then the dose required (greater than 100uM) to inhibit 50% of the enzyme activity. You need to increase you concentration or it will be better to make different doses starting from 100 uM as mentioned by Angel Gaudioso
I am wondering if that concentration does harm the cell lines or not because I do not know if IC50 that is mentioned is for Biomedical or cellular assay?
IC50 is not a great readout because it is dependent on the concentration of the substrate (assuming we are discussing a competitive inhibitor). It is described by:
IC50 = Ki * (1 + S/Km)
where Ki is the dissociation constant of the inhibitor, S is the substrate concentration and km is a constant that describes the substrate concentration at which enzyme activity is half of its maximum. So you can see that depending on the substrate concentration you can have different IC50 values.
Most people have already commented that to see significant inhibition under conditions that gave a 100uM IC50, you would need to use concentrations of your inhibitor that are around 100uM, but if that is not possible, you can try reducing the substrate concentration, which would lower the IC50 and lower the concentration of inhibitor that you would need to see inhibition.
Thanks everyone's answer. I will change the concentration and apply to see which one works
I typically did experiments where I was trying to identify the effective dose of a drug in killing cancer cells or bacteria.
In this case, one wants to use a culture of cells that is in log phase, in other words, it is a active cell culture that is dividing at a standard rate. In order to achieve this, you want to be changing your cells every 1-3 days to have them in good shape. Then, the day before you do your experiment, split the cells and let them grow overnight.
Then, I would use a dilution strategy. OFten it seemed convenient to use a microtiter plate with the cells in each well except for one row (or column) with only media.
You can put 75 ul (microliter) of cells or media in each well. Then, use a concentration of drug at 2x time the initial concentration. Take 75 ul of your drug mixed in media and mix it with column 1 (gently please) by pipetting carefully until mixed. I like to use a multi microtiter pipette to do an entire row.
Then, take 75 ul of cells/drug out of column 1 and mix into column 2, giving you a 1:2 dilution; the next column is a 1:4 dilution, 1:8, etc. Don't mix into the last column which is the media.
Then, you need to have a read out. For living cells, it actually works well to take the microtiter plate and put it right into the microplate reader. Measure the densities of the cells. Subtract the densities of the cells from the media alone to get the cell concentration. The advantage of this method is that you can use the same plate for serial readings on separate days. The disadvantage is that dead cells also have a density (see below for a discussion on this).
The growing cells will increase in density and by plotting the growth curves vs. the drug concentration, you can determine the concentration of drug that inhibits the growth by 50%. The IC50 will give you an estimate of the drug concentration that is required for the effect that you wish to find. Of course, killing 50% of cancer cells doesn't do much good for a patient since it will take about one day for those cells to grow up again!
A better way, supposedly, is to use viability agents that detect whether cells are viable or not. When working with AML cells, I got the same results using these agents as using the concentration of growing cells, but that might not work for all systems: you need to check. See my papers on leukemia cells for an example.
I did not really answer your question properly.
If the IC 50 is >100 ul, this means that 50% of the effect will be inhibited at a concentration of more than 100 ul. The fact that they said the inhibitor works at a concentration of more than 100 ul suggests that the inhibitor is not a very powerful inhibitor. You might wish to look for an inhibitor with an IC50 in the range of less than 20 ul or so unless this is not relevant to your question. Once you get up to 100 ul, it is more likely to affect other components of your system.
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