10 replies to this topic

[hidayahhh]

this is example of data collected from elisa plate reader. i dont know hoe to plot the graf of % cell viability vs log concentration

Attached Files

•  hFOB P1.xls   3.23K   113 downloads

[madelingirly]

I will say what I would do if I were u
for % of viability I calculate it as abs of test divide by abs of control (no treatment) X100
(get average of Abs for test repeats in ur exp / by average abs of repeats of control)
Each experiment should be repeated 2-3 times at different days or more, so I would calculate STD from my these repeats ( if I do the experiment 3 times) I would calculate % of vaibility for each time as previously mentioned  then calculate standard deviation or Standard error for these 3 times,
the i guess X axis would be log conc, and Y axis would be % of viability..
This I would do if I were u

[Ms VV]

Hello,

I would also appreciate some help with this.  I do MTS assays to measure the activity of certain compounds.  I do each trial with 4 replicates for each group and then normalize the abs of each well with the average abs of the control.  After that I calculate the average and standard deviation for each group.  I then repeated the experiment 2 more times for a total of three trials, but now I end up with three averages (each having a standard deviation) and I can't figure out the proper way to calculate the final average and standard deviation.  I have tried various search terms in google and could not find anything that match exactly with what I need.  

For example, the data that I got for drug A at a certain concentration is (data are % of control):  Trial 1: mean = 90 , SD= 5; Trial 2: mean = 94, SD = 4; Trial 3: mean = 89, SD = 3.  How do I calculate the overall mean and standard deviation of these values?

I was told that I can just propagate the errors of each trial.  Is this OK to do?

It would be a lot easier if I could just average the abs for each treatment group, normalize to the control and calculate the average and SD of the three trials, but what happens to the error associated with the abs of the replicated wells?

Would greatly appreciate a response if anyone has any experience with this type of calculation.

Thanks!

[DRT]

Ms VV, on 05 March 2013 - 05:47 PM, said:

I do each trial with 4 replicates for each group and then normalize the abs of each well with the average abs of the control.  After that I calculate the average and standard deviation for each group.  I then repeated the experiment 2 more times for a total of three trials, but now I end up with three averages (each having a standard deviation) and I can't figure out the proper way to calculate the final average and standard deviation.

Arrange the data into an ANOVA table this will separate the error into within-trial and between-trial components (many text books use the term blocks to represent the different trials you performed).
  Chances are that there will be no significant difference between the trials and you will be able to treat them as a single average and standard deviation as you suggested but it never hurts to check.

[Ms VV]

Hi DRT, thank you for responding! Just to clarify, your suggestion is to enter the % viability of each well for each trial and do an ANOVA to test for statistical significant differences?  So I should enter the data as follows?

Trial 1         Trial 2          Trial 3   
% well 1      % well 1        % well 1
% well 2      % well 2       % well 2   
% well 3      % well 3       % well 3   
% well 4      % well 4        % well 4

If there are no significant differences, then I can just use all the values to calculate one average and standard deviation?

Edited by Ms VV, 06 March 2013 - 10:35 PM.

[DRT]

That seems correct, most software will want the results in a single column with a tag denoting which trial in an adjacent column.

good luck

[Peniel]

Ms VV, on 05 March 2013 - 05:47 PM, said:

Hello,

I would also appreciate some help with this.  I do MTS assays to measure the activity of certain compounds.  I do each trial with 4 replicates for each group and then normalize the abs of each well with the average abs of the control.  After that I calculate the average and standard deviation for each group.  I then repeated the experiment 2 more times for a total of three trials, but now I end up with three averages (each having a standard deviation) and I can't figure out the proper way to calculate the final average and standard deviation.  I have tried various search terms in google and could not find anything that match exactly with what I need.  

For example, the data that I got for drug A at a certain concentration is (data are % of control):  Trial 1: mean = 90 , SD= 5; Trial 2: mean = 94, SD = 4; Trial 3: mean = 89, SD = 3.  How do I calculate the overall mean and standard deviation of these values?

I was told that I can just propagate the errors of each trial.  Is this OK to do?

It would be a lot easier if I could just average the abs for each treatment group, normalize to the control and calculate the average and SD of the three trials, but what happens to the error associated with the abs of the replicated wells?

Would greatly appreciate a response if anyone has any experience with this type of calculation.

Thanks!

Hi Ms W,

Just a quick question. When you do your MTS assay do you use 96wells plates. If yes, do you change the media everyday. I find out that in 72hrs the media only wells are almost empty. I would love any tips as this is my first time with this type of experiment.  Thanks

[Ms VV]

Thanks DRT!  I'll try that and see.

Hi Peniel!  Yes, I use 96 well plates.  I usually incubate for 48 h without replacing the medium and have no noticeable decrease in volume by looking at the wells.  There is a decrease of 0.1-0.2 mL when measured.  I incubated a few times for  72 h and also did not noticed a big decrease in volume.  I use 0.1 mL per well.  The wells could hold up to 0.3 mL, maybe you could use a higher volume if you are not already doing so.  Sorry, I don't really have any suggestions other since I haven't had that issue.

[DRT]

Peniel, on 08 March 2013 - 03:01 AM, said:

Hi Ms W,

Just a quick question. When you do your MTS assay do you use 96wells plates. If yes, do you change the media everyday. I find out that in 72hrs the media only wells are almost empty. I would love any tips as this is my first time with this type of experiment.  Thanks

A problem with the humidity levels in the incubator?
Are all your media controls in a row along one side? Be wary of always running your experiments in the same direction across a plate. In theory it shouldn't make a difference and all wells are identical but there is always the potential for biasing the results.

[Tabaluga]

DRT, on 10 March 2013 - 10:57 AM, said:

A problem with the humidity levels in the incubator?
Are all your media controls in a row along one side? Be wary of always running your experiments in the same direction across a plate. In theory it shouldn't make a difference and all wells are identical but there is always the potential for biasing the results.

and keep in mind the "edge effect" (higher evaporation at the edges of well plates)

[Peniel]

DRT, on 10 March 2013 - 10:57 AM, said:

Peniel, on 08 March 2013 - 03:01 AM, said:

Hi Ms W,

Just a quick question. When you do your MTS assay do you use 96wells plates. If yes, do you change the media everyday. I find out that in 72hrs the media only wells are almost empty. I would love any tips as this is my first time with this type of experiment.  Thanks

A problem with the humidity levels in the incubator?
Are all your media controls in a row along one side? Be wary of always running your experiments in the same direction across a plate. In theory it shouldn't make a difference and all wells are identical but there is always the potential for biasing the results.

I don't know if the humidity level is a problem. Yeah I run my experiment in a column (B2-G2) for a drug concentration and B3-G3..... The media is usually just to fill up the empty wells.

Do you think having the wells low on media could affect my result? I assumed the plate reader only take A1(ddH20) into consideration whilst generating absorbances.