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 entries
Run 1: Ht31/Ht31P D9/D12
Related topics
20.109(S08):Testing_cell_viability_(Day3)
|
Viability staining
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