If you have the data and want to calculate the IC50, then it's just like calculating a half-life. Select the data, insert a "scatter plot," and set the Y-axis to "logarithmic." It should be approximately linear over some range. Select "add trend line" and pick "exponential." Then display equation and you've got it. I attached an example.

I found that using the Add-On features can be helpful:

Excel add-in

ED50V10 (Readme) is an Excel add-in for calculating IC50/EC50 values. Input your data in the left columns, and your results will be shown in the right half of the Excel table.

To calculate IC50, input 50 in the "INTERPOLATE..." table (highlighted in blue), the result will be shown on the right (highlighted in green).

For log-transformation, go to Data Transformation on the upper-right, input 3 in the DOSE (X-axis)/1st cell.

Four-Parameter Logistic Function

Four-parameter (A, B, C, D) logistic function (or Sigmoidal)

is frequently used to fit dose-response curves. The drug data x is in logarithmic form. This is a standard function in most statistics software. The parameter C is the estimate of IC50 or EC50.

I found this on the sciencegateway.org website.  Hope that helps.

If you can get the add-in XLfit from IDBS, it has many useful capabilities, including calculating IC50s and drawing the IC50 curves.

 Thanks, Dr Bendot. Which is IC50 dose in the example?

To calculate the IC50 value: The inhibitor concentration  against the percent activity is plotted ([I]-Activity % graph).  Using the linear (y=mx+n) or parabolic (y=ax2+bx+c) equation on  this graph for y=50 value x point becomes IC50 value. 

Dr Bendot, you did not illustrate which of these is the Ic50 value. Is the y value the IC50?and how would it be represented as it appears as e.g. 134. 55e-0.002x

I believe if someone has clear understanding of what IC50 ( half maximal inhibitory concentration) basically infers: the concentration of extract which hunt 50 of under investigation radicals either of DPPH or ABTS. So, it does not matter which software you are using, you have plot a scatter plot of various extract concentration (normally from 2-2000ug/ml), add a trend line, see the equation of line, like Y=2.358X+4.69;. Now substitute y=0.5 in this equation to calculate the x (IC 50)

Personally, I do not prefer the linear fit method suggested by Muhammad Mushtaq. I know that some people use this method, but it has a few disadvantages compared with nonlinear regression to a suitable equation, such as the Hill equation. (1) The linear method forces you to truncate the data set of % inhibition versus inhibitor concentration values. This is due to the fact that a graph of these values is not linear, although it may appear linear over a narrow range. It requires an arbitrary judgement of which points to include and which to exclude. (2) There is usually some scatter in the data points. Scatter within the subset of points that is used in the linear calculation will introduce error into the calculation, especially if outliers are included in the subset. The nonlinear regression method is less sensitive to scatter because it uses all of the data, and obvious outliers can be excluded while still allowing the data to be fit.

Can somebody help me how to calculate my IC50 value in GraphPad Prism? I have my absorbance values and my percentage data.

I also have my NPs MTT result. Can somebody tell me how to analyze this data and calculate IC50?

Thanks

To calculate the IC50 value: The inhibitor concentration  against the percent activity is plotted ([I]-Activity % graph).  Using the linear (y=mx+n) or parabolic (y=ax2+bx+c) equation on  this graph for y=50 value x point becomes IC50 value. 

Kunal Pal, this time you copied and pasted Hatibe Ertürk Kara's answer. Is there any chance that you could offer an original thought?

i got different IC50 values using different sources like linear regression graph(trendline,eq in chart y=mx+c),graph pad prism,ic50 calculator from AAT Bioquest (internet). now i am confused which is the correct one. can any one check from the file i have attached which is the correct answer

i am sorry because instead of giving answer i have asked a new question

I think the problem is that you are allowing the minimum and maximum % inhibition values to float, rather than fixing them at 0% and 100%, respectively. The programs are fitting with a minimum % inhibition greater than zero and a maximum % inhibition less than 100, resulting in the obviously incorrect IC50 of about 100. Do not use linear regression (see my comment above on that subject). Go back to Prism and set the minimum and maximum % inhibition at 0 and 100, respectively. As far as I can tell, AAT Bioquests calculator does not allow you to make that change. You should get a value close to 320 µg/ml.

I agree with Adam. The minimum and maximum % inhibition values have to be set at 0 and 100 respectively and the linear regression also has a problem of scatter. I also agree with Muhammad and Ayam up to inserting the Trendline. However, after this, the R2 should be checked for the goodness-of-fit of the data to the Trendline for a linear model. Change the Trendline to those for non-linear models like the exponential, logarithmic and polynomial, each time observing the R2. The Trendline with the largest R2 is usually the one from which I determine the IC50 in Excel.

IC50 curves follow a particular mathematical function. They are not linear, exponential, polynomial, or logarithmic. These fits should not be used. Excel does not have the actual function that should be used in the trendline feature, which is the Hill equation. This represent a square hyperbola, like the Michaelis-Menten equation or Langmuir binding isotherm equation, but adds a slope factor (the Hill coefficient) to allow for negative or positive cooperativity of binding.

The Hill equation is present in the Excel add-in called XLfit as equation 205, but this requires a purchase. Other commercial nonlinear regression programs in common use such as Graphpad Prism and SigmaPlot can also be used. I believe the full 4-parameter Hill equation is the basis for the IC50 fitting in some free on-line resources. Unfortunately, the ones I looked at did not allow the user to fix the upper and lower bounds at 100 and 0, which can result in incorrect values for IC50.

By the way, it is not necessary to use the logarithms of the inhibitor concentrations when fitting IC50 curves, which then requires a logistic form of the Hill equation for the fitting. However, it is useful to plot IC50 curves on a logarithmic x-axis when the inhibitor concentrations cover multiple orders of magnitude. The y-axis should then be % inhibition or % control activity, since it is not possible to plot the control value on the graph (because there is no logarithm of zero).

Also by the way, when testing novel inhibitors, it is not uncommon to observe that the % inhibition doesn't approach 100%. Sometimes inhibition even starts to decrease at high inhibitor concentrations. This is often caused by artifacts in the experiment. One common artifact is insolubility of the compound. It is just not soluble enough to reach a concentration at which 100% inhibition would occur. In such cases, only the data at lower concentrations should be used. Another common artifact is that the compound interferes with the assay system in some way. It is important to identify such problems and not blindly try to fit an IC50 curve to problematic data by using ever more complex mathematical equations that have no basis in reality.

Dr Adam is very correct. The Hill equation is most appropriate for determination of IC50 because the minimum and maximum effects appear very clearly in the equation. I think this therefore should help: I = Io + ImaxC^a/IC50^a + C^a. where I is the predicted effect of the inhibitor, Io, the effect of inhibitor concentration 0, Imax is its maximum effect, C is the inhibitor concentration at time t, IC50 is the inhibitor concentration for which 50% of maximum effect is obtained and a is the Hill coefficient of sigmoidicity. In pharmacology, the unit of inhibitor effect, I depends on the response considered; the unit of inhibitor concentration C and IC50 is usually expressed in mol/L or g/L, and a has no unit. This is a general four-parameter Hill equation and is suitable for modeling both positive or negative responses (i.e. the observed response may increase or decrease with increasing concentrations).

It seems everyone is having issue for this IC50. Just simple and use Graphpad prism. All are solved easily and you are done!

thanks jamail Lu i got it

Is this IC50 value calculated using excel valid.? If valid then please mention such type of research articles..

IC50 can be calculated by excel too but complicated way and you will get approximate only and in some cases, your results will be wrong. Noted that IC50 is calculated non-linear regression analysis (the curve is Sigmoidal curve).

Easier way is just use Graphpad prism, you are done with all.

Some people use GraphPad but they use wrongly because they look for interception at 50 from their various doses of cell viability % and this is wrong. Use appropriate way of finding IC50. Read manual of GraphPad and do or attend training for it.

But can you go back to the file that Pushpa Reddy share?

I prefer excel result base on the data that she has.

Because 50% close to what excel result than GraphPad or AAT Bioquest .

Excel provides non-linear regression also that we can check which regression that show the best or the closest one to our scatter result data in graph.

Excel does not calculate EC50 automatically, but you may do some calculations by yourself, e.g. by using Hill formula and solver. Here is my template I use for EC50 value calculation in cytotoxicity experiments. I am sure, you may apply different formulas and make your own template. Good luck!

You can search how to calculate IC50 online or textbook, and then you can set up the formulation in Excel though insert function!

both method of Dr. Shabir Ahmed and Professor Vima Petrikaite is right i copy the data from Professor Vilma and put in the calculator of Dr. Ahmed the results were the same so the method of Dr. Shabir is more easy

This is a bit of a late response, but we have indeed received several questions similar to what was initially discussed by Pushpa Reddy and Adam Shapiro. This is in regards to our online regression model tool not restricting response values between a set minimum and maximum limit. Traditionally, four parameter logistic regression does not have a set minimum or maximum, but is contingent upon the inputted experimental data. That being said, often, it does not make coherent sense biologically speaking for a response value to fall into the negative. In these cases, one can either utilize a probit/logit type regression or specifically set the minimum limit as Adam suggested. For example, the minimum response limit can be set to zero. This would reduce the four parameter logistic regression to a three parameter logistic regression.

As I mentioned, we have received several inquiries about such a function and have worked to integrate this function into our online IC50 calculator. To utilize this feature, select “Set minimum response to zero (0)" after data entry. This should address the concern Pushpa Reddy initially raised. If there are any follow up questions or suggestions, feel free to contact us for support.

https://www.aatbio.com/tools/#data-analysis-and-regression

Thank you so much Dr Jennifer for the response

Prism Graphpad would be a optimal software for LC 50 calculation.

Try this one

Hi Junapoudi,

You did not use the inhibition % in your calculations?

Can you give some details about your attached xlsx file?

Thanks,

Hi Iman

I given insect direct formal in xlsx file

direct add you values in xlsx file give % Inhibition once try it

All the best

You need more than 5 points to make a trend line, simulate the equation, and calculate the IC50 according to the equation.

I think you must use graph pad prism, excel will not be the appropriate choice for this. All the best

I think using of prism soft ware is better and easier for calculation of IC50.

www.malariaresearch.eu/eumalar/sites/.../IC50%20Calc.xls

To calculate the IC50 value: The inhibitor concentration(in ppm or microgram/ml)  against the percent activity is plotted .  Using the linear (y=mx+n) or non linear regression equation (y=ax2+bx+c) equation on  this graph for y=50 value x point becomes IC50 value.  solve for x using quadratic formula

x=(-b±√(b2-4ac))/2a

https://www.youtube.com/watch?v=CD9CZjzDTEE

I'm afraid excel is not the appropriate software to calculate IC50. I recommend GraphPad prism. It's super easy to work with and you will have a more precise answer.

I don't have graph pad but I have sigma plot. How can I use sigma plot to calculate IC50

Hi Cosma,

You can try our IC50 calculator from following link. Simply copy and paste the data from your Excel, and click the "process data". Hope it helps. Thanks!

https://www.aatbio.com/tools/ic50-calculator

Best regards,

Jennifer

With SigmaPlot, you can calculate IC50 using the 4-parameter Hill equation.

I use graph pad prism for this and it really works well

Graph pad work just fine

The websites ic50.tk and ic50.org do the calculations online, the latter gives better quality plots!

Fantastic method for quality IC50 plot, including enzyme kinetic plot. What about double reciprocal plot (1/Vo versus 1/[S]. Do they work the same way?

Thank you. The fitting is done directly to the untransformed data by non-linear least squares, so it should be more accurate than doing a linear transformation, such as the 1/v versus 1/[S] plot.

For IC50 calculation my all time user friednly software is Graphpad prism.

DPPH inhibition (%)={(A0 –A1)/A0}×100 where A0 is the absorbance of control and A1 is the absorbance of test.

In DPPH free radical scavenging method, IC50 (Half maximal Inhibitory Concentration) value is the concentration of the sample that could scavenge 50% of DPPH free radical. You can calculate from the equation.

Rather than excel use this online tool IC50 Calculator | AAT Bioquest!!

Is there anyone used Igor pro8 software for calculating IC50 for enzyme inhibition and what type of fitting used?

Graphpad helps you to go for regression analysis and put 50 value for unknown and it will give you the concentration as predicted.

Are there any one use Spotfire for IC50 of inhibitors? Thanks

The best software for this is GraphPad Prism.

https://www.helsinki.fi/en/unitube/video/9f872c04-0128-4952-82a0-d22a649e9fa6

I calculated IC50 of concentration over percentage inhibition on Excel and I got a Y= MX-C in stead of Y=MX+C. For each of the 5 different concentration of my sample, there was a triplicate determination of percentage inhibition. How do I go about the IC50 on Excel.

Happy Easter, Dr Onyedikachi. I just saw your question here on ResearchGate. You have done well by having triplicate for each concentration and have calculated the % inhibition. Again, the equation of the graph is already derived. It is likely that your line graph cut through the negative X or Y-axis. Ensure your R2 is very strong (>0.95). To calculate the IC50 value, from the equation of the graph that you got (Y = MX-C), change Y to 50 (50= MX-C) then make X the subject of the formula. The X value should be your IC50 value.

This video is a Instructions for plotting the data from cell viability assays in Excel can help you calculate it.

https://www2.helsinki.fi/en/unitube/video/9f872c04-0128-4952-82a0-d22a649e9fa6

Important question... Good

Good question

https://www.youtube.com/watch?v=ZrefBJs-C1g

https://www.youtube.com/watch?v=ZrefBJs-C1g

Please watch this video for details about calculation of IC50 using excel

Nice to see good replies.....is graph prism necessary or we can do that on excel sheet

Good question

https://www.youtube.com/watch?v=fMghQw2Ry2c This video also explains better.

Dear all,

I've just tried different ways to calculate also EC50.

If you want to try, I share my modest contribution : https://www.researchgate.net/post/How_to_calculate_an_EC50_for_a_bioassay_eg_LEIA_EHIA_inhibition_test

I hope it will help some people like us.

Don't hesitate to complete on what I have written.

Any comments, any suggestions, I will take it.

Thanks in advance.

Use this https://www.aatbio.com/tools/ic50-calculator

Thank you so much

Thanks for Graph pad Prism to get accurate results

It is possible to calculate IC50 using Excel. The basic equation for IC50 is IC50 = -log10 (x/C), where x is the concentration of your compound and C is the concentration of your inhibitor. To calculate IC50, you will need to plot a graph of the response of your compound to the inhibitor on a logarithmic scale. Once the graph is plotted, you can use the Excel ‘slope’ function to calculate the slope of the line. Then, you can use the equation to calculate IC50 from the slope of the line.

The IC50 (inhibitory concentration) is the concentration of a drug or other inhibitor that is required for 50% inhibition of a given biological or biochemical function. Using Excel, you can calculate the IC50 by using the formula =LOG(C5/B5)/LOG(2) where C5 and B5 are the concentrations of the drug or inhibitor that result in 50% inhibition and 100% inhibition, respectively.