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Alamar Blue Assay


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#1 cwong1215

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Posted 23 May 2009 - 11:25 PM

Hi Guys,

I'm going to be doing some compound screening using the alamar blue proliferation assay in a 96 well plate format on either HEK or MCF-7 cells. Does anyone have a good written protocol that they follow and can share?

I've seen two wavelengths given for the readings, one in the 600s and the other in the 500s. Does it matter which is used? Also it may seem a bit naive but is it pretty common to run a no cell (medium only) control and use it to normalize the other samples? ie: setting the no cell control to a value of one by dividing by itself then dividing all other values by that number as well.

Thanks!

#2 pito

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Posted 24 May 2009 - 10:32 AM

Hi Guys,

I'm going to be doing some compound screening using the alamar blue proliferation assay in a 96 well plate format on either HEK or MCF-7 cells. Does anyone have a good written protocol that they follow and can share?

I've seen two wavelengths given for the readings, one in the 600s and the other in the 500s. Does it matter which is used? Also it may seem a bit naive but is it pretty common to run a no cell (medium only) control and use it to normalize the other samples? ie: setting the no cell control to a value of one by dividing by itself then dividing all other values by that number as well.

Thanks!


Maybe you should read the file in the attachment.
I have used the method before, however not for the things you want to use it for.
I do not have a lot of time at the moment, being very bussy. But if this file does not help, just let me know and I might be able to help a bit more.

And yes you need to use both wavelengths.
And yes you need to run a no cell test.

This is all explained in the file, if I am not mistaken.
I have however noticed that this "newer" online file is smaller then the one I have.
I have a protocol text of about 19 pages , this file is only 11. I hope it does help a bit.

Attached Files


If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.

#3 cwong1215

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Posted 24 May 2009 - 06:30 PM

Thanks Pito,

A question for you, sorry if this seems a small issue, I would just like to make sure that the experimental design is sound.

If for example I was running a time course experiment looking at the effect of a compound at 6, 12, 24, 48 and 72 hours and had determined that the optimal incubation period for the alamar blue was 2 hours. Would it be correct to add the reagent at 4, 10, 22, 46 and 70 hours and then read the plate at the end of the 2 hour incubation period?

Edited by cwong1215, 24 May 2009 - 06:31 PM.


#4 pito

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Posted 25 May 2009 - 08:27 AM

Thanks Pito,

A question for you, sorry if this seems a small issue, I would just like to make sure that the experimental design is sound.

If for example I was running a time course experiment looking at the effect of a compound at 6, 12, 24, 48 and 72 hours and had determined that the optimal incubation period for the alamar blue was 2 hours. Would it be correct to add the reagent at 4, 10, 22, 46 and 70 hours and then read the plate at the end of the 2 hour incubation period?



I am not 100% sure what you mean.

You mean that after doing the standard alamar blue test you get the results and it show that you best use a 2 hours incubation period.
And then you want to check your cells after they are 6 , 12, 24, 48 and 72 hours old ?

if this is the case, then yes.

However I never used it in such short periods.
I used the alamar blue to determine the growht (survival) of a certain fungus during a few months.
I never used such short time intervals.
Besides that my optimal incubation time was something about 2 days...

Its been a while since I used it, but I hope this helps.
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.




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