User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/02/17

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Viability staining Main project page
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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
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)



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