Cell lab diary

AlamarBlue assay

10.08.2013-14.08.2013

Trial alamarBlue assay for HeLa

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- HeLa

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.

This are the results of a measurement of absorbance:

We've counted an average for each concentration and gained such results:

After that, we've counted % of cell growth by dividing an average for each time and concentration by appropriate average for the control. The results are shown here:

So the graphs serve as the summary:

We've decided to conduct each experiment twice so here are the results from the second 96-well plate:

We've counted an average for each concentration and gained such results:

After that, we've counted % of cell growth by dividing an average for each time and concentration by appropriate average for the control. The results are shown here:

So the graphs serve as the summary:

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

AlamarBlue assay for 143b

In order to examine possible cytotoxic effect of acrylamide on bone tissue, we've conducted AlamarBlue assay for 143b cell line. Basing on the protocol obtained for HeLa cell line and after conducting trial assay, we've decided to incubate cells for 24h at 37°C prior to addition of acrylamide. We’ve prepared two 96-well plates with 5000 cells per each well for three times. We've measured cytotoxity of higher acrylamide concentrations i.e. 1-5mM as well as lower: 0,2-1mM.

Here we would like to present the outcomes of our experiment.

24.08.2013-29.08.2013

26.08.2013-31.08.2013

28.08.2013-03.09.2013

Intravital observation

After hours spent on observing the cells and the changes that they've been undergoing we've decided to share some photos from our bright field microscope. We've prepared two 6-wells plates both for HeLa and 143b cell lines: 200.000 cells/well, incubation for 24 hours, adding acrylamide in concentrations 1,2,3,4 and 5mM. Here we present the examples from our intravital observation.

24 hours incubation

Negative control	1mM concentration
2mM concentration	3mM concentration
4mM concentration	5mM concentration

48 hours incubation

Negative control	1mM concentration
2mM concentration	3mM concentration
4mM concentration	5mM concentration