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:
- fMLP starting at 0.5 uM; threefold dilutions
- Anti-IgE starting at 10 ug/mL; threefold dilutions
- Anti-IgE row, now in Ca free conditions (EDTA 5 mM in RPMI, final conc. 2.5 mM)
- Abs panel:
- CD63 PE (Lot: 64074)
- CD123 PE-Cy5 (Lot: 65847)
- CD203c PE-Cy7 (Lot: 02, Exp: 2011-01-22)
- 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).