Shreffler:Notebook/Alex Daily/2010/06/02

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R03 - CaFree (Experiment #5)

  • Obtained buffy coat from Tuesday (6/1) drawing.
  • Going back to 1X DHR (used 8X in past few experiments)
    • 8X added 96 uL 5mM DHR solution to 6 mL total blood+rpmi, now back to 12uL
  • Can eliminate DMSO rows.
  • Experiment row layout:
    1. RPMI
    2. fMLP starting at 0.5 uM; threefold dilutions
    3. Anti-IgE starting at 10 ug/mL; threefold dilutions
    4. Anti-IgE row, now in Ca free conditions (EDTA 5 mM in RPMI, final conc. 2.5 mM)
  • Abs panel:
    1. CD63 PE (Lot: 64074)
    2. CD123 PE-Cy5 (Lot: 65847)
    3. CD203c PE-Cy7 (Lot: 02, Exp: 2011-01-22)
    4. HLA DR APC (Lot: 65678)


Tried a couple things:

  • Used cold PBS after stim step to halt degranulation
  • Drew higher blood volume into pipettes to compensate for viscosity (blood retention on sides of pipette tips).


Carbo (004 ADB)

  • Past experiments used IgE at 10 ug/mL (30 uL from aliquot into 270 uL RPMI), which is supraoptimal. Changed IgE concentration to 1 ug/mL.
  • Also began using IL-3 in fMLP/IgE samples (past experiments did not).


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