Good day everyone,
I am starting cell proliferation assay using alamarBlue and according to the protocol I have, 100% reduced form of alamarBlue is used as positive control. My question is, if I want to measure the fluorescence on different time points, should I use different positive control or can I use the same one (e.g. I measured the positive control at 24h along with the samples and use that to calculate the % AB reduction on 48h)?
I am still learning about this, so all the advice will be really appreciated!
If you are measuring all your test samples after incubating them overnight with almar blue then you should do the same with your control as well. If you have multiple replicates of each samples you measure at different timepoints then you would need corresponding replicates of control for each time point. In whatever way you are measuring your test samples, measure the control in the same way. A good control is one that is exposed to every single condition your test is exposed to except for the thing you are testing for.
I totally agree with Praveesh. I would like to add that it is quite a sensitive test and you can expected a lot of variability. I suggest you have a negative control, a positive control, a blank one (only media) and a another blank (empty well. At least on the first trial just to have an idea about how is the absorbtion/ reflection of your plate and its homogeneity). And use a culture plate with black wall and transparent bottom (ideal for fluorescence reading tests). I used to run those in a 96 well plate and had sextuplicate of treatments (min 6 well per treatment, per condition, per time point). And as mentioned before, each time point should be taken as a different experiment. At the beginning, when you are standardizing the experiment, you could read the same plate in different time points to identify the ideal period of incubation with AB. Once you find it, you stick with this and vary only your treatment/ condition. For example: 12 hours drug treatment (6 hours AB incubation), 24 hours treatment (6 hours AB incubation), 36 hours treatment (6 hours AB incubation), and so on and so forth. I used it for viability, so my time points might be a bit shorter than yours. Good luck!
The alamar blue or resazurin is a dye used to quantify the percentage of viable or inhibited cells by the mitochondrial dehydrogenase enzyme. The positive control is your untreated samples(only cells) that you will remain blue after the 3-4hrs of incubation. However the treated samples will turn to pink due to the formation of resorufin complex.
Thank you Praveesh Valissery Taís Adelita and Michele S Majoumouo. So if I get this right, I have to incubate the sample for the 100% reduced form at the same time before autoclave it? I checked the protocol from BioRad (https://www.bio-rad-antibodies.com/measuring-cytotoxicity-proliferation-spectrophotometry-fluorescence-alamarblue.html) and it just mentions that to make 100% reduced form of alamarBlue, is to autoclave it with media (1:10 ratio), there's no mention of incubation along with the sample. So I am a little confused about this.
Hi Atika. I used this test for cytotoxicity in neural stem cells. My positive control was Staurosporine and here is an outline of how I proceeded:
I used the Resazurin sodium salt from Sigma and I prepared a 20X stock solution (PM=251.17. Then 20X solution is aprox 880 micromolar – or 0.88 milimolar) by dissolving 4.4mg of salt in 20mL of PBS, filtered sterilized (0.22um) and kept it at 4C.
To perform the experiment, I diluted the salt at 1X final concentration in my culture media (with Pen/Strep but no supplements) and replaced all the media from each well of the 96 well plate with 200uL of this fresh media with resazurin (my cells had been previously treated for 12, 24 or 48 hours with different drugs and controls). After the color started to visually change (6 hours of resazurin incubation on my case. You can imagine that this time depends on the metabolic activity of your cells. If you leave it too long, all salt will be metabolized = all wells will be equally purple), I transferred 190uL of media from each well to my “reading plate” (96 well plate with transparent bottom and black wall that I reuse a few times after properly wash) and proceeded to the fluorescence reader machine.
I hope this is helpful. Good luck!
Taís Adelita
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