Team:Warsaw/Notebook

From 2013.igem.org

(Difference between revisions)
(→AlamarBlue assay)
(→18.08.2013-23.08.2013)
Line 566: Line 566:
|3271010
|3271010
|2082594
|2082594
-
|1639722
+
|1639722
 +
|1978809
|1191210
|1191210
|1191003
|1191003
Line 616: Line 617:
|-
|-
|24h
|24h
-
|7142615,5
+
|4556384,5
-
|7263264,5
+
|5096861,5
-
|5376427,5
+
|4836618,5
-
|4907832
+
|4293681,75
-
|4004682,25
+
|3789115,5
-
|4113004,25
+
|3521978
|-
|-
|48h
|48h
-
|8838765
+
|6989439,25
-
|8129346,25
+
|7047779,25
-
|5803169
+
|4917687
-
|4254053,75
+
|3805363,5
-
|2478317
+
|2355179,75
-
|2251420
+
|2287818,75
|-
|-
|72h
|72h
-
|10505847,75
+
|6494556,75
-
|10123624,5
+
|5994983,5
-
|4601891
+
|3309743,75
-
|2715412,75
+
|1899441,25
-
|2027336,5
+
|1199548,25
-
|1522866,5
+
|1161574,5
|-
|-
|96h
|96h
-
|8986261,25
+
|9660892,75
-
|5455715,5
+
|6665392,75
-
|1441368,25
+
|1730496,25
-
|1088298,5
+
|1120537
-
|1199471
+
|1251568
-
|1121372,5
+
|1086199,75
|-
|-
|}
|}
Line 663: Line 664:
|-
|-
|24h
|24h
-
|1,02
+
|1,12
-
|0,75
+
1,06
-
|0,69
+
0,94
-
|0,56
+
0,83
-
|0,57
+
0,77
|-
|-
|48h
|48h
-
|0,92
+
|1,01
-
|0,66
+
0,7
-
|0,48
+
0,54
-
|0,28
+
0,34
-
|0,25
+
0,33
|-
|-
|72h
|72h
-
|0,96
+
|0,92
-
|0,44
+
|0,51
-
|0,26
+
|0,29
-
|0,19
+
|0,18
-
|0,14
+
|0,18
|-
|-
|96h
|96h
-
|0,61
+
|0,69
-
|0,16
+
|0,18
|0,12
|0,12
|0,13
|0,13
-
|0,12
+
|0,11
|-
|-
|}
|}

Revision as of 07:22, 9 September 2013

Notebook

Contents

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:

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:

caption


caption

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:

caption


caption

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 2
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

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


72hours incubation

Negative control

1mM concentration

2mM concentration

3mM concentration

4mM concentration

5mM concentration



Retrieved from "http://2013.igem.org/Team:Warsaw/Notebook"