Cell lab diary

AlamarBlue assay

10.08.2013-14.08.2013

Trial alamarBlue assay

In order to obtain repeatable and reliable results of experiment we’ve decided to conduct trial assay to determine plating density and concentration of acrylamide. In order to examine how HeLa cell line would react to certain acrylamide concentrations we’ve implemented test assay providing 0,5mM, 1mM and 5mM concentrations. First of all we’ve harvested cells and prepared different densities: 2500, 5000, 7500 and 10000 cell per well of 96-well plate. After adding alamarBlue to each well in amount of 1/10 of the culture volume we’ve measured fluorescence after 24 hours incubation.

According to obtained results we’ve decided to use the density of 5000 cells per well. Secondly, we’ve conducted test assay with three different acrylamide concentration in order to establish the scope of our research. The initial results were as follows:

• 0,5mM an 1 mM concentration -> most of the cells are viable
• 5mM concentration -> most of the cells dead

Taking the outcome into account we’ve decided to examine 1mM, 2mM, 3mM, 4mM and 5mM acrylamide concentration for 24h, 48h, 72h, and 96h.

16.08.2013-21.08.2013

alamarBlue assay

Following the protocol we checked cytotoxity of acrylamide for HeLa cells. We’ve decided to implement two different ways of incubating the cells: 1st adding cell suspention directly to certain acrylamide concentration 2nd adding cell suspention to the wells and incubating them for 24h at 37°C, then adding certain acrylamide concentration.

17.08.2013-22.08.2013

We’ve decided that the second type of measurement i.e. incubating cells for 24h at 37°C prior to addition of acrylamide will better reflect optimal conditions of cell growth. We’ve prepared two 96-well plates with 5000 cells per each well and incubated them for 24 hours. After this time we’ve added acrylamide according to the protocol.

18.08.2013-23.08.2013

In order to confirm the results and state the receptiveness of the test, we’ve decided to prepare two more 96-well plates in identical conditions.

Alamar Blue results for HeLa