Team:Warsaw/Notebook
From 2013.igem.org
Notebook
Contents |
Cell lab diary
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 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.
So this 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've 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'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:
% 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 the summary:
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:
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've 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'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:
% 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 the summary:
We've 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've 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'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:
% 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 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 another 96-well plates in identical conditions.
This are the results of a measurement 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've 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'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:
% 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 the summary:
Alamar Blue assay for HeLa 0,2-1mM
As in some probes we've 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've 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 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.
Cell growth assay
As we wanted to bring under scrutiny the results obtained from AlamarBlue assay we've 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 cells division (so the cytostatic effect of acrylamide) or by cells death (so the cytotoxic effect of acrylamide).
First of all we've 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've prepared 6-wells plates for HeLa, HEK293 and 143b cell lines. After 24 hours incubation we've added acrylamide in concentrations: 1,3 and 5mM for HeLa and HEK and 1,2 and 5mM for 143b. We've conducted the assay three times and juxtaposed the results.
We've 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, live 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 | 7,5 | ||||
1mM | 1.400.000 | 10,3 | ||||
3mM | 664.000 | 9,6 | ||||
5mM | 300.000 | 25,1 |
HeLa cell growth 72h | ||||||
---|---|---|---|---|---|---|
Acrylamide concentration | live cells number | percent of dead cells | ||||
NC | ||||||
1mM | ||||||
3mM | ||||||
5mM |
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 |
HeLa cell growth 72h | ||||||
---|---|---|---|---|---|---|
Acrylamide concentration | live cells number | percent of dead cells | ||||
NC | ||||||
1mM | ||||||
3mM | ||||||
5mM |
Results for 143b
HeLa cell growth 24h | ||||||
---|---|---|---|---|---|---|
Acrylamide concentration | live cells number | percent of dead cells | ||||
NC | ||||||
1mM | ||||||
3mM | ||||||
5mM |
HEKcell growth 48h | ||||||
---|---|---|---|---|---|---|
Acrylamide concentration | live cells number | percent of dead cells | ||||
NC | ||||||
1mM | ||||||
3mM | ||||||
5mM |
HeLa cell growth 72h | ||||||
---|---|---|---|---|---|---|
Acrylamide concentration | live cells number | percent of dead cells | ||||
NC | ||||||
1mM | ||||||
3mM | ||||||
5mM |