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

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* Measure in Wallace 1420 Victor 2TM, excitation 544 nm emission 390 nm
* Measure in Wallace 1420 Victor 2TM, excitation 544 nm emission 390 nm
* This shows the metabolic (mitochondria) activity of cells
* This shows the metabolic (mitochondria) activity of cells
==Results==
==Results==
*{{todo| 1 D9 (P21)}}
*{{todo| 1 D9 (P21)}}

Revision as of 08:15, 17 February 2010

<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
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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

  • 1 D9 (P21)
  • {{todo| 1 D12 (P27)}
  • 2 D9 (P21)
  • {{todo| 2 D12 (P27)}
  • 3 D9 (P21)
  • 3 D12 (P27)

Conclusion

  • DONE Ow..

Related topics

20.109(S08):Testing_cell_viability_(Day3)



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