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Viability staining
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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
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01
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02
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03
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04
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05
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06
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07
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08
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09
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10
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11
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12
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A
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1A1
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1A2
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1A3
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1A4
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1A5
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1A6
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2A1
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2A2
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2A3
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2A4
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2A5
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2A6
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B
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1B1
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1B2
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1B3
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1B4
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1B5
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1B6
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2B1
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2B2
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2B3
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2B4
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2B5
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2B6
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C
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1C1
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1C2
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1C3
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1C4
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1C5
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1C6
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2C1
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2C2
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2C3
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2C4
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2C5
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2C6
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D
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1D1
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1D2
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1D3
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1D4
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1D5
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1D6
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2D1
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2D2
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2D3
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2D4
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2D5
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2D6
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E
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3A1
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3A2
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3A3
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3A4
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3A5
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3A6
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|
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F
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3B1
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3B2
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3B3
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3B4
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3B5
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3B6
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G
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3C1
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3C2
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3C3
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3C4
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3C5
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3C6
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H
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3D1
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3D2
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3D3
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3D4
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3D5
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3D6
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- 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
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1 HT31P
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2 HT31P
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3 HT31P
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4 HT31
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5 HT31
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6 HT31
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A (CTR)
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1824
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1982
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1953
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5125
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5172
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5939
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B (CSE)
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956
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964
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785
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4465
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5006
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5815
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C (cse +8p)
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1075
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1104
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971
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5017
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5241
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5526
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D (cse+6Bnz)
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761
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674
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645
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4408
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5385
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4943
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D9 (passage 21)
#2
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1 HT31P
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2 HT31P
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3 HT31P
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4 HT31
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5 HT31
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6 HT31
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A (CTR)
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2232
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2560
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2506
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6431
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6394
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7264
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B (CSE)
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1113
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1050
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998
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6077
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6513
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7171
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C (cse +8p)
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1224
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1024
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1234
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5606
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6203
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7559
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D (cse+6Bnz)
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730
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826
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858
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5309
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4856
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2518
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D9 (passage 21)
#3
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1 HT31P
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2 HT31P
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3 HT31P
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4 HT31
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5 HT31
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6 HT31
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A (CTR)
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2359
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2494
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2434
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5865
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6713
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6843
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B (CSE)
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998
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929
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957
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5129
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5632
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6518
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C (cse +8p)
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946
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1033
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1026
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5562
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5622
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6125
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D (cse+6Bnz)
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940
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1123
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982
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6428
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5590
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4116
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D12 (passage 27)
#1
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1 HT31P
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2 HT31P
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3 HT31P
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4 HT31
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5 HT31
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6 HT31
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A (CTR)
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2151
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2049
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2021
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7501
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7411
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7507
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B (CSE)
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773
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848
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814
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5447
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6630
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6700
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C (cse +8p)
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718
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769
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769
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4324
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4835
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6349
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D (cse+6Bnz)
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877
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874
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1002
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5387
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7649
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6039
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D12 (passage 27)
#2
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1 HT31P
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2 HT31P
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3 HT31P
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4 HT31
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5 HT31
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6 HT31
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A (CTR)
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1389
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2124
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1980
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7432
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7069
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7977
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B (CSE)
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925
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1021
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972
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4957
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5760
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6746
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C (cse +8p)
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960
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1030
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1069
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5381
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4675
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6522
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D (cse+6Bnz)
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767
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877
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1138
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5216
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6143
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6754
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D12 (passage 27)
#3
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1 HT31P
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2 HT31P
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3 HT31P
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4 HT31
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5 HT31
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6 HT31
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A (CTR)
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2071
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2158
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2331
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7252
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7248
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7881
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B (CSE)
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1098
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953
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1087
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5078
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5458
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5958
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C (cse +8p)
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926
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1324
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1106
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4599
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4899
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5503
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D (cse+6Bnz)
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908
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738
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957
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4963
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4311
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6101
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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)
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