Team:Warsaw/Cellular biology lab journal

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===AlamarBlue assay for 143b===
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===AlamarBlue assay for 143b 1-5mM===
In order to examine possible cytotoxic effect of acrylamide on bone tissue, we have conducted AlamarBlue assay for 143b cell line.
In order to examine possible cytotoxic effect of acrylamide on bone tissue, we have conducted AlamarBlue assay for 143b cell line.
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Here we would like to present the outcomes of our experiment.
Here we would like to present the outcomes of our experiment.
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====24.08.2013-29.08.2013====
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====Flourescence measurement for 143b cells (29.08.2013):====
 
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Revision as of 21:41, 4 October 2013

Cellular biology lab journal

Contents


This is the journal of our cellular biology lab. Click here for our genetic lab journal.

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

The results are summarized below:

caption


caption

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

The results are summarized below:

caption


caption

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

The results are summarized below:

caption


caption

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

The results are summarized below:

caption


caption

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

The results are summarized below::

caption caption

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

The results are summarized below::

caption caption

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

The results are summarized below::

caption caption

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

The results are summarized below::

caption caption

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

The results are summarized below::

caption caption

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

The results are summarized below::

caption caption

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

The results are summarized below::

caption caption

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

The results are summarized below::

caption caption

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:

750px caption

AlamarBlue assay for 143b 1-5mM

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

caption

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 intended 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 number 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

caption

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

caption

Caspase-Glo 3/7 Assay

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

Caspase activity after 24hours

caption

Caspase activity after 48hours

caption

Cell Cycle

As we wanted to state how acrylamide may influence cell cycle we've decided to implement Cell Cycle Assay. This method employs flow cytometry in order to envision phases of the cell cycle. Before analysis, we'd permeabilised cells with cold ethanol and treated them with propidium iodide. PI binds to DNA quantitatively so the intensity of the fluorescence of the stained cells is correlated with the amount of DNA that they contain.

We've prepared two probes for each incubation time and acrylamide concentration for each of those lines: HeLa, HEK293 and 143B. The results are presented below:

24 hours incubation time

caption

caption

caption


48 hours incubation time

caption

caption

caption

Histograms for Cell Cycle assay

HeLa cell line upper row: 24h incubation bottom row: 48h incubation acrylamide concentrations from left: NC, 1mM, 3mM, 5mM

143B cell line upper row: 24h incubation bottom row: 48h incubation acrylamide concentrations from left: NC, 1mM, 2mM, 5mM

HEK293 cell line upper row: 24h incubation bottom row: 48h incubation acrylamide concentrations from left: NC, 1mM, 3mM, 5mM

Flow Cytometry

Results for Cell Cycle assay

HeLa cell line upper row: 24h incubation bottom row: 48h incubation acrylamide concentrations from left: NC, 1mM, 3mM, 5mM

143B cell line upper row: 24h incubation bottom row: 48h incubation acrylamide concentrations from left: NC, 1mM, 2mM, 5mM

HEK293 cell line upper row: 24h incubation bottom row: 48h incubation acrylamide concentrations from left: NC, 1mM, 3mM, 5mM

caption


Cisplatin control

HeLa cell line upper row: 24h incubation bottom row: 48h incubation acrylamide concentrations from left: NC, 1mM, 3mM, 5mM

143B cell line upper row: 24h incubation bottom row: 48h incubation acrylamide concentrations from left: NC, 1mM, 2mM, 5mM

HEK293 cell line upper row: 24h incubation bottom row: 48h incubation acrylamide concentrations from left: NC, 1mM, 3mM, 5mM

caption

caption

caption



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