Team:Warsaw/Notebook

From 2013.igem.org

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Revision as of 19:29, 29 August 2013

Notebook

Contents

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

Measurement of fluorescence
Time NC 1mM 2mM 3mM 4mM 5mM
24h 6700039 7271548 6745967 6666035 5047406 3616931 4274714 4841184 4253892 3779748 4584429 3307109
6990006 7608869 7866854 7774202 6314529 6526844 4860078 5655352 3865177 4119912 4800916 3759563
48h 7459856 9994235 7075587 8043466 5643349 5769546 3223686 3660815 2360577 2224518 2110235 1911433
8919429 8981540 9176248 8222084 6474175 5325606 5477827 4653887 2967761 2360412 2903491 2080521
72h 11364152 10673740 10631207 10630185 5019728 4835926 2621560 2821185 2289734 2032174 1577851 1468166
10984789 9000710 10211232 9021874 3789425 4762485 3372217 2046689 1966275 1821163 1463407 1582042
96h 9979086 8726507 6938730 6794761 1608486 2085391 1177176 1057910 1101076 1330276 1329184 890891
6677357 10562095 3926716 4162655 1014022 1057574 1024019 1094089 1045779 1320753 1254068 1011347
% cell growth
Time NC 1mM 2mM 3mM 4mM 5mM
24h 1,02 0,75 0,69 0,56 0,57
48h 0,92 0,66 0,48 0,28 0,25
72h 0,96 0,44 0,26 0,19 0,14
96h 0,61 0,16 0,12 0,13 0,12



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