Dear Sameer,

The following is the required protocol:

Alamar Blue Assay

The alamar blue assay (AB)  was carried out according to manufacturer’s instructions. Briefly, control medium was removed; the cells were rinsed with PBS and 2 mL of an AB solution (5% [v/v] solution of AB dye) prepared in fresh medium (without FBS or supplements) were added to each well. Following 3 hours incubation, AB fluorescence was quantified at the respective excitation and emission wavelength of 540 and 595nm using a Tecan Genios microplate reader. The results were averaged over 3 different independent experiments (n=3, each conducted with one week interval) with 3 replicates per experiment (3 x 6 well

plates), each replicate being prepared from different T75 flasks in order to take into

account the biological variability. Finally, for each plate the reading was also done in triplicate (values obtained from 3 different wells averaged) in order include the technical variability due to the efficiency of AB assay, sensitivity of the plate reader or simply related to the sample preparation. For each experiment, wells containing only the AB solution without cells were also prepared and incubated for 3h. The fluorescence measured in those was used as a background and subtracted. However, although this protocol is routinely used in the literature, some variations in the AB solution concentration and exposure time have been made purposely throughout this study. These modifications will be clearly highlighted and discussed at the appropriate points in the manuscript, for clarity. Again, each experiment was conducted in triplicates (3 x 6 well plates) which have been prepared from different flasks in order to have independent replicate based on different cell populations.

For more on this topic, please use the following link:

http://arrow.dit.ie/cgi/viewcontent.cgi?article=1081&context=nanolart

Hoping this will be helpful,

Rafik

Rafik Karaman Sir, Thanks for your reply. Is it necessary to use a known drug as a control in in-vitro cytotoxicity and anticancer activity assay by alamar blue assay.   

Dear Sameer,

Yes, for sure. This is needed for potency's comparisons.

Rafik

Sir, for different cell line we have to choose a different different control drugs or should I use an only one drug as a control for all the chosen cell lines.

Dear Sameer,

There are different types of cancers and each one is better investigated with a certain cell line. Therefore, for each cell line you need to use different drug as a control. Herein some of the cancer types and their corresponding cell lines:  Lung (A549, A-427), kidney (A498), Leukemia (K-562, MOLT-4, REH, Nalm-6 etc.), breast cancer (MCF-7, MDA-MB etc.), cervical (Hela),  and etc. and each one has its drug's control.

Please note, that you should compare the activity to normal cells as well.

Rafik

OK Sir,

This gave me very crystal clear idea about this type of assay. From where I would get a normal cell lines? And if I am doing a study on liver cancerous cell line, so for that whether I required to check an effect on normal liver cell line (non-cancerous) or any other normal cell line will also work?

Dear Sameer,

Normal liver and any other cells you can buy, for example, please see the following link:

 http://www.innoprot.com/en_productos_subfamilias.asp?idsf=19&id=8&gclid=Cj0KEQjw-YO7BRDwi6Stp7T296ABEiQAD6iWMWmySi5UDhFW9H_1LtGRd7UXLNlqGObruzzb3svtTxUaAgH38P8HAQ

Every test on any cancerous cells should be compared with the corresonding normal cells.

Rafik