Shreffler:Notebook/Alex Daily/2010/04/28

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Lab-drawn Whole Blood Test

  • Today's experiment will not use buffy coat; instead, we will draw blood from lab members (my experiment will use Bert's blood).
  • Experiment will span two days:
    1. First day will be a simplified DHR experiment (no NAC). Two sets, one in usual medium (RPMI), one in RPMI with 5mM EDTA (to chelate Ca).
      • (Next step of RO3 is to determine if ROS production is Ca dependent, kinase dependent, ...)
    2. Each set will consist of three series of three tubes each:
      1. RPMI
      2. fMLP starting at 0.5 uM; threefold dilutions
      3. Anti-IgE starting at 10 ug/mL; threefold dilutions
    3. Also, abs panel will be slightly altered; Wayne wants to try gating with CRTH2 (CD294)
      1. CD294 AlexaFluor 647
      2. CD63 FITC
      3. CD203c PE
      4. HLA DR PE-Cy7
    4. Second day, also two sets, both in RPMI; one set in polystyrene tubes and one set in polypropylene tubes
  • This experiment allows us to observe: difference between regular stimulation in presence/absence of Ca, difference between different tubes (activated basophils may have slight tendency to stick more to polystyrene tubes), and finally, time-dependence of experiment in regards to blood draw time (fresh vs day-old blood).


  1. Double wash cells as usual
  2. Incubate with DHR as usual
  3. Pipette cells into stim tubes here is step to use EDTA RPMI
  4. Stain with abs cocktail
  5. FACS Lyse

Notes

  • Stim step was prolonged by 10 minutes (so 30 minutes total).

RX Project

  • Today we are running the first real sample, this time with both cisplatin and carboplatin (test run from two weeks ago only tested carboplatin). Some things to remember:
    1. Use 50 uL cold PBS after stimulation step in order to slow/arrest degranulation.
    2. Use staining buffer when making abs cocktail.
    3. For highest concentrations of stimulant, dilute stock tenfold(???), so Carboplatin has final highest concentration of (10 mg/mL)/10 = 1 mg/mL, and Cisplatin (1 mg/mL)/10 = 0.1 mg/mL (final concentration is half this, since this concentration is mixed ~1:1 with blood).
    4. Stock IL-3 is 10 ug/mL, 5x protocol's assumed 2 ug/mL, so make sure to dilute five-fold first.
    5. Use fMLP and Anti-IgE at optimal concentrations, so fMLP at top concentration

Note: only used 100 uL of Abs cocktail per sample rather than 110 uL; also, mistakenly added Abs to "A: Unstained." Finally, aspirated a little bit of "H: Carb 3."


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