<html><style type="text/css">
.todo { color: red }
.done { color: green}
</style></html>
 Viability staining
|
<html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>
|
Summary
- The supernatant of 3 24-wells plates per donor are put on 1 96-wells plate
Materials & Methods
Materials
Method
Preparations ELISA
- Take 200 μL of supernatant of each well and put it in 96-wells plate
- Divide like shown below, (#plate (24-wells);horizontal lane; vertical lane)
96-wells plate
|
|
01
|
02
|
03
|
04
|
05
|
06
|
07
|
08
|
09
|
10
|
11
|
12
|
| A
|
1A1
|
1A2
|
1A3
|
1A4
|
1A5
|
1A6
|
2A1
|
2A2
|
2A3
|
2A4
|
2A5
|
2A6
|
| B
|
1B1
|
1B2
|
1B3
|
1B4
|
1B5
|
1B6
|
2B1
|
2B2
|
2B3
|
2B4
|
2B5
|
2B6
|
| C
|
1C1
|
1C2
|
1C3
|
1C4
|
1C5
|
1C6
|
2C1
|
2C2
|
2C3
|
2C4
|
2C5
|
2C6
|
| D
|
1D1
|
1D2
|
1D3
|
1D4
|
1D5
|
1D6
|
2D1
|
2D2
|
2D3
|
2D4
|
2D5
|
2D6
|
| E
|
3A1
|
3A2
|
3A3
|
3A4
|
3A5
|
3A6
|
|
|
|
|
|
|
| F
|
3B1
|
3B2
|
3B3
|
3B4
|
3B5
|
3B6
|
|
|
|
|
|
|
| G
|
3C1
|
3C2
|
3C3
|
3C4
|
3C5
|
3C6
|
|
|
|
|
|
|
| H
|
3D1
|
3D2
|
3D3
|
3D4
|
3D5
|
3D6
|
|
|
|
|
|
|
- Supernatant is stored @ -20 °C and used for ELISA later on
- Remaining cells are used for viability staining. (See Alamar Blue staining)
Alamar Blue staining
- Remove remaining medium from 24-wells plates
- Wash twice with PBS
- Add per well 475 μL HBSS + 25 μL Alamar blue
- For ~75 wells that's 35.625 mL HBSS + 1875 μL Alamar blue
- Incubate ~30 min. 37 °C
- Measure in Wallace 1420 Victor 2TM, excitation 544 nm emission 390 nm
- This shows the metabolic (mitochondria) activity of cells
Results
Alamar Blue
D9 (passage 21)
| #1
|
1
HT31P
|
2
HT31P
|
3
HT31P
|
4
HT31
|
5
HT31
|
6
HT31
|
| A (CTR)
|
1824
|
1982
|
1953
|
5125
|
5172
|
5939
|
| B (CSE)
|
956
|
964
|
785
|
4465
|
5006
|
5815
|
| C (cse +8p)
|
1075
|
1104
|
971
|
5017
|
5241
|
5526
|
| D (cse+6Bnz)
|
761
|
674
|
645
|
4408
|
5385
|
4943
|
D9 (passage 21)
| #2
|
1
HT31P
|
2
HT31P
|
3
HT31P
|
4
HT31
|
5
HT31
|
6
HT31
|
| A (CTR)
|
2232
|
2560
|
2506
|
6431
|
6394
|
7264
|
| B (CSE)
|
1113
|
1050
|
998
|
6077
|
6513
|
7171
|
| C (cse +8p)
|
1224
|
1024
|
1234
|
5606
|
6203
|
7559
|
| D (cse+6Bnz)
|
730
|
826
|
858
|
5309
|
4856
|
2518
|
D9 (passage 21)
| #3
|
1
HT31P
|
2
HT31P
|
3
HT31P
|
4
HT31
|
5
HT31
|
6
HT31
|
| A (CTR)
|
2359
|
2494
|
2434
|
5865
|
6713
|
6843
|
| B (CSE)
|
998
|
929
|
957
|
5129
|
5632
|
6518
|
| C (cse +8p)
|
946
|
1033
|
1026
|
5562
|
5622
|
6125
|
| D (cse+6Bnz)
|
940
|
1123
|
982
|
6428
|
5590
|
4116
|
D12 (passage 27)
| #1
|
1
HT31P
|
2
HT31P
|
3
HT31P
|
4
HT31
|
5
HT31
|
6
HT31
|
| A (CTR)
|
2151
|
2049
|
2021
|
7501
|
7411
|
7507
|
| B (CSE)
|
773
|
848
|
814
|
5447
|
6630
|
6700
|
| C (cse +8p)
|
718
|
769
|
769
|
4324
|
4835
|
6349
|
| D (cse+6Bnz)
|
877
|
874
|
1002
|
5387
|
7649
|
6039
|
D12 (passage 27)
| #2
|
1
HT31P
|
2
HT31P
|
3
HT31P
|
4
HT31
|
5
HT31
|
6
HT31
|
| A (CTR)
|
1389
|
2124
|
1980
|
7432
|
7069
|
7977
|
| B (CSE)
|
925
|
1021
|
972
|
4957
|
5760
|
6746
|
| C (cse +8p)
|
960
|
1030
|
1069
|
5381
|
4675
|
6522
|
| D (cse+6Bnz)
|
767
|
877
|
1138
|
5216
|
6143
|
6754
|
D12 (passage 27)
| #3
|
1
HT31P
|
2
HT31P
|
3
HT31P
|
4
HT31
|
5
HT31
|
6
HT31
|
| A (CTR)
|
2071
|
2158
|
2331
|
7252
|
7248
|
7881
|
| B (CSE)
|
1098
|
953
|
1087
|
5078
|
5458
|
5958
|
| C (cse +8p)
|
926
|
1324
|
1106
|
4599
|
4899
|
5503
|
| D (cse+6Bnz)
|
908
|
738
|
957
|
4963
|
4311
|
6101
|
Conclusion
Samples should have been been into the wells at random to prevent the cells to concentrate in the first samples.
Related
Related topics
20.109(S08):Testing_cell_viability_(Day3)
|
Viability staining
