Contents 1 AlamarBlue assay for HeLA 1-5mM 1.1 10.08.2013-14.08.2013 1.2 16.08.2013-21.08.2013 1.3 17.08.2013-22.08.2013 1.4 18.08.2013-23.08.2013 2 Alamar Blue assay for HeLa 0,2-1mM 2.1 29.08.2013-03.09.2013 2.2 31.08.2013-05.09.2013 2.3 02.09.2013-07.09.2013 3 AlamarBlue assay for HEK293 1-5mM 3.1 16.08.2013-21.08.2013 3.2 18.08.2013-22.08.2013 3.3 20.08.2013-25.08.2013 4 AlamarBlue assay for HEK293 0,2-1mM 4.1 16.08.2013-21.08.2013 4.2 18.08.2013-22.08.2013 4.3 20.08.2013-25.08.2013 5 AlamarBlue assay for 143b 5.1 24.08.2013-29.08.2013 5.2 26.08.2013-31.08.2013 5.3 28.08.2013-03.09.2013 6 Intravital observation 7 Cell growth assay 7.1 Results for HeLa 7.2 Results for HEK293 7.3 Results for 143b 8 Caspase-Glo 3/7 Assay 8.1 Caspase activity after 24hours 8.2 Caspase activity after 48hours

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AlamarBlue assay for HeLA 1-5mM

10.08.2013-14.08.2013

Trial alamarBlue assay for HeLa

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

According to obtained results we have decided to use the density of 5000 cells per well. Secondly, we have conducted test assays 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 are dead

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

16.08.2013-21.08.2013

AlamarBlue assay- HeLa

Following the protocol we checked the cytotoxity of acrylamide for HeLa cells. We have 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.

Below are the results of a measurement of absorbance for the plate with prior incubation:

Measurement of fluorescence 2
Time	NC		1mM		2mM		3mM		4mM		5mM
24h	7373758	8272076	7192444	7424543	6641569	5650021	4952094	5922398	5436664	5306241	3556654	4163111
	7173972	6545080	7188951	6915072	6116307	6441105	5747208	4019806	5217586	5387212	4096686	3533518
48h	21498976	9060887	9766205	10285037	6448099	6495998	3563610	3727306	3257840	2889223	2154377	1595037
	7589281	10161667	9724553	10407614	6814538	6072379	1263710	4840663	3767542	3464691	1186634	2151884
72h	10194118	11427411	10016065	8593429	4926555	4852670	2535034	4609394	1841320	1914045	870765	943721
	9116994	10566937	8968114	10328330	5798232	4700243	2421617	2200734	1854485	1719428	995642	705623
96h	10551875	12256688	8642510	8263400	2534417	2322236	1096577	1883518	1711689	1695785	899049	806117
	1645895	9714010	5134617	5176732	1108646	1045172	788164	1062518	1399573	1454092	853618	739065

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

Average of measurement
Time	NC	1mM	2mM	3mM	4mM	5mM
24h	7341221,5	7180252,5	6212250,5	5160376,5	5336925,75	3837492,25
48h	12077702,75	10045852,25	6457753,5	3348822,25	3344824	1771983
72h	10326365	9476484,5	5069425	2941694,75	1832319,5	878937,75
96h	8542117	6804314,75	1752617,75	1207694,25	1565284,75	824462,25

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

% cell growth
Time	1mM	2mM	3mM	4mM	5mM
24h	0,978073268	0,84621483	0,70293159	0,726980619	0,522732116
48h	0,831768463	0,534683924	0,277273114	0,27694207	0,146715235
72h	0,917697999	0,490920571	0,284872242	0,1774409	0,08511589
96h	0,796560706	0,2051737	0,141381141	0,183243188	0,096517321

So the graphs serve as summary:

17.08.2013-22.08.2013

We have 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 have prepared two 96-well plates with 5000 cells per each well and incubated them for 24 hours. After this time we added acrylamide according to the protocol.

This are the results of measurements of absorbance:

Measurement of fluorescence 2
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

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

Average of measurement
Time	NC	1mM	2mM	3mM	4mM	5mM
24h	7142615,5	7263264,5	5376427,5	4907832	4004682,25	4113004,25
48h	8838765	8129346,25	5803169	4254053,75	2478317	2251420
72h	10505847,75	10123624,5	4601891	2715412,75	2027336,5	1522866,5
96h	8986261,25	5455715,5	1441368,25	1088298,5	1199471	1121372,5

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

% cell growth
Time	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

So the graphs serve as summary:

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

Measurement of fluorescence 2b
Time	NC		1mM		2mM		3mM		4mM		5mM
24h	5689442	5477740	5369019	6121442	3874536	2893257	4184202	3790779	3042292	3374958	3285526	3225528
	6372624	7876223	7325975	5727235	6427409	4842799	6249360	5321887	3410938	4275630	3259977	4842161
48h	10639247	8148331	8915964	10177195	4871060	5010788	4367575	4642340	3085628	2590736	3532060	2466736
	10957449	10453297	11004473	8473231	5160946	4336928	4824239	4789970	2504349	2232280	3357123	2379384
72h	10283165 12000635	9635513	8880168	3811216	4058437	3236952	3193475	1630107	1478097	1569754	1804201
	10766834	11976578	10027043	9133051	4402895	4117753	3321155	2998722	1708259	2018056	1787657	1582990
96h	11739077	13027189	9808090	9472783	2524300	2334036	1189210	1055404	1300283	1628039	1353530	1310116
	9625754	12521745	6479618	5128707	1327488	1099976	1015119	974386	1378368	1532597	1409938	1110367

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

Average of measurement
Time	NC	1mM	2mM	3mM	4mM	5mM
24h	6354007,25	6135917,75	4509500,25	4886557	3525954,5	3653298
48h	10049581	9642715,75	4844930,5	4656031	2603248,25	2933825,75
72h	11256803	9418943,75	4097575,25	3187576	1708629,75	1686150,5
96h	11728441,25	7722299,5	1821450	1058529,75	1459821,75	1295987,75

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

% cell growth
Time	1mM	2mM	3mM	4mM	5mM
24h	0,97	0,71	0,77	0,56	0,57
48h	0,96	0,48	0,46	0,26	0,29
72h	0,84	0,36	0,28	0,152	0,149
96h	0,66	0,16	0,09	0,12	0,11

So the graphs serve as summary:

18.08.2013-23.08.2013

In order to confirm the results and state the receptiveness of the test, we have decided to repeat the experiments by testing another 96-well plates in identical conditions.

Those are the results of measurements of absorbance:

Measurement of fluorescence 3
Time	NC		1mM		2mM		3mM		4mM		5mM
24h	3974421	4463736	5292334	5060967	4153885	4370635	3999981	3517207	3499234	3388296	2859389	3571247
	4797393	4989988	5183920	4850225	5442555	5379399	4915546	4741993	4079843	4189089	4221010	3436266
48h	6503529	6944856	5589520	7406073	4893204	4810250	3282289	3640323	2292748	2648757	2909252	1695347
	8529450	5979922	7483886	7711638	4212594	5754700	4821335	3477507	1932753	2546461	2868302	1678374
72h	5854635	9116820	6242532	6355495	3973902	3911469	2009124	1970110	1083848	1332132	1259200	1059008
	5723413	5283359	5546619	5835288	3271010	2082594	1639722	1978809	1191210	1191003	1302859	1025231
96h	8331318	9814058	7669153	7555556	1926251	1977639	1303301	1170557	1286656	1326686	1249020	1097188
	4114415	16383780	5936493	5500369	1118274	1899821	1009362	998928	1154602	1238328	1197251	801340

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

Average of measurement
Time	NC	1mM	2mM	3mM	4mM	5mM
24h	4556384,5	5096861,5	4836618,5	4293681,75	3789115,5	3521978
48h	6989439,25	7047779,25	4917687	3805363,5	2355179,75	2287818,75
72h	6494556,75	5994983,5	3309743,75	1899441,25	1199548,25	1161574,5
96h	9660892,75	6665392,75	1730496,25	1120537	1251568	1086199,75

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

% cell growth
Time	1mM	2mM	3mM	4mM	5mM
24h	1,12	1,06	0,94	0,83	0,77
48h	1,01	0,7	0,54	0,34	0,33
72h	0,92	0,51	0,29	0,18	0,18
96h	0,69	0,18	0,12	0,13	0,11

So the graphs serve as summary:

Alamar Blue assay for HeLa 0,2-1mM

As in some probes we have observed growth of viability for lower acrylamide concentration, we decided to implement AlamarBlue assay also for 0,2 0,4 0,6 0,8 and 1mM concentrations. We have conducted the experiment for three times according to protocol and the results are as follows:

29.08.2013-03.09.2013

% cell growth
Time	1mM	2mM	3mM	4mM	5mM
24h	0,89	0,92	0,96	0,93	0,83
48h	0,81	0,73	0,6	0,66	0,54
72h	0,83	0,73	0,72	0,51	0,6
96h	0,77	0,68	0,67	0,64	0,5

Graph summary:

31.08.2013-05.09.2013

% cell growth
Time	1mM	2mM	3mM	4mM	5mM
24h	0,96	0,85	0,87	0,94	0,82
48h	0,94	0,74	0,77	0,78	0,66
72h	0,99	0,73	0,79	0,77	0,62
96h	0,86	0,64	0,66	0,74	0,39

Graph summary:

02.09.2013-07.09.2013

% cell growth
Time	1mM	2mM	3mM	4mM	5mM
24h	0,99	1,01	1,22	0,99	0,8
48h	0,92	0,8	0,76	0,67	0,6
72h	0,87	0,74	0,68	0,6	0,28
96h	0,89	0,79	0,75	0,53	0,16

Graph summary:

AlamarBlue assay for HEK293 1-5mM

In order to examine possible cytotoxic effect of acrylamide on kidney, we have conducted AlamarBlue assay for HEK293 cell line. Basing on the protocol obtained for HeLa cell line and after conducting trial assays, we have decided to incubate cells for 24h at 37°C prior to addition of acrylamide. We have prepared two 96-well plates with 5000 cells per each well for three times. We have measured the cytotoxity of higher acrylamide concentrations i.e. 1-5mM as well as lower: 0,2-1mM.

16.08.2013-21.08.2013

% cell growth
Time	1mM	2mM	3mM	4mM	5mM
24h	0,51	0,44	0,35	0,27	0,18
48h	0,62	0,31	0,25	0,15	0,12
72h	0,38	0,16	0,12	0,08	0,07
96h	0,125	0,086	0,075	0,069	0,066

Graph summary:

18.08.2013-22.08.2013

% cell growth
Time	1mM	2mM	3mM	4mM	5mM
24h	0,78	0,71	0,62	0,29	0,25
48h	0,53	0,33	0,17	0,12	0,1
72h	0,33	0,18	0,13	0,09	0,08
96h	0,09	0,06	0,05	0,048	0,047

Graph summary:

20.08.2013-25.08.2013

% cell growth
Time	1mM	2mM	3mM	4mM	5mM
24h	0,81	0,69	0,38	0,36	0,21
48h	0,66	0,34	0,2	0,15	0,1
72h	0,29	0,15	0,09	0,075	0,063
96h	0,12	0,07	0,07	0,06	0,055

Graph summary:

AlamarBlue assay for HEK293 0,2-1mM

16.08.2013-21.08.2013

% cell growth
Time	1mM	2mM	3mM	4mM	5mM
24h	0,95	0,91	0,82	0,7	0,62
48h	0,88	0,94	0,9	0,89	0,79
72h	0,5	0,43	0,45	0,55	0,4
96h	0,61	0,63	0,56	0,5	0,2

Graph summary:

18.08.2013-22.08.2013

% cell growth
Time	1mM	2mM	3mM	4mM	5mM
24h	0,9	0,82	0,9	0,91	0,8
48h	0,8	0,76	0,75	0,68	0,65
72h	0,72	0,72	0,67	0,62	0,53
96h	0,63	0,53	0,5	0,52	0,27

Graph summary:

20.08.2013-25.08.2013

% cell growth
Time	1mM	2mM	3mM	4mM	5mM
24h	0,89	0,6	0,61	0,58	0,52
48h	0,63	0,58	0,57	0,56	0,48
72h	0,65	0,63	0,62	0,56	0,54
96h	0,57	0,54	0,52	0,5	0,38

Graph summary:

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AlamarBlue assay for 143b

In order to examine possible cytotoxic effect of acrylamide on bone tissue, we have conducted AlamarBlue assay for 143b cell line. Basing on the protocol obtained for HeLa cell line and after conducting trial assays, we have decided to incubate cells for 24h at 37°C prior to addition of acrylamide. We have prepared two 96-well plates with 5000 cells per each well for three times. We have measured the 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 have been undergoing we have decided to share some photos from our bright field microscope. We have prepared two 6-wells plates both for HeLa and 143b cell lines: 200.000 cells/well, incubated 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

72hours incubation

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

96hours incubation

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

Cell growth assay

As we wanted to bring under scrutiny the results obtained from AlamarBlue assay we have decided to carry out the cell growth assay. This would enable us to state whether the loss of cell viability, that we have observed, was caused by decrease in cell division (so the cytostatic effect of acrylamide) or by cell death (so the cytotoxic effect of acrylamide).

First of all we have carried out preliminary assay to lay down the adequate cells amount per well. The results show that the cells reached proper confluence for the following concentrations:

• 300.000 cells per well for HeLa
• 100.000 cells per well for 143b
• 600.000 cells per well for HEK293

We have prepared 6-wells plates for HeLa, HEK293 and 143b cell lines. After 24 hours incubation we added acrylamide in following concentrations: 1,3 and 5mM for HeLa and HEK and 1,2 and 5mM for 143b. We have conducted the assay three times and juxtaposed the results.

We have counted cell number after 24, 48 and 72 hour according to the protocol. Usage of Trypan Blue additionally enabled us to count the amount of dead cells and find the proportion between acrylamide concentration, alive and dead cells.

So the results are as follows:

Results for HeLa

HeLa cell growth 24h
Acrylamide concentration	live cells number	percent of dead cells
NC	1.368.000	0,6
1mM	1.124.000	3,2
3mM	814.000	7,1
5mM	716.000	14,2

HeLa cell growth 48h
Acrylamide concentration	live cells number	percent of dead cells
NC	2.200.000	2,8
1mM	1.400.000	7,4
3mM	664.000	29
5mM	300.000	85,4

HeLa cell growth 72h
Acrylamide concentration	live cells number	percent of dead cells
NC	3.848.000	3,4
1mM	2.255.200	9,7
3mM	354.160	182
5mM	234.360	240

Results for HEK293

HEK293 cell growth 24h
Acrylamide concentration	live cells number	percent of dead cells
NC	2.255.000	2
1mM	1.756.000	5,4
3mM	1.273.000	21
5mM	536.250	50

HEKcell growth 48h
Acrylamide concentration	live cells number	percent of dead cells
NC	4.096.354	2.5
1mM	2.461.000	5.1
3mM	1.091.104	29.4
5mM	489.583	85.6

HEK cell growth 72h
Acrylamide concentration	live cells number	percent of dead cells
NC	4.583.200	3,05
1mM	2.598.800	10,2
3mM	531.000	188
5mM	316.000	277

Results for 143b

143b cell growth 24h
Acrylamide concentration	live cells number	percent of dead cells
NC
1mM
3mM
5mM

143b cell growth 48h
Acrylamide concentration	live cells number	percent of dead cells
NC
1mM
3mM
5mM

143b cell growth 72h
Acrylamide concentration	live cells number	percent of dead cells
NC
1mM
3mM
5mM

Caspase-Glo 3/7 Assay

In order to examine whether there occurs cell apoptosis, we've decided to implement Caspase-Glo 3/7 assay obtained from Promega. This test enabled us to show caspse activity in cells treated with acrylamide in comparison to control. The results are as follows:

Caspase activity after 24hours

Caspase activity after 48hours