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

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Viability staining
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |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

 * 96-wells plates
 * HBSS
 * PBS

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)


 * 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

Alamar Blue
     

Conclusion
Samples should have been been into the wells at random to prevent the cells to concentrate in the first samples.

Run 1: Ht31/Ht31P D9/D12

 * Splitting cells 10Feb2010
 * Counting cells 12Feb2010
 * Putting to S0 15Feb2010
 * Stimulating cells 16Feb2010

Related topics
20.109(S08):Testing_cell_viability_(Day3)


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