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:
 * 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, ...)
 * Each set will consist of three series of three tubes each:
 * RPMI
 * fMLP starting at 0.5 uM; threefold dilutions
 * Anti-IgE starting at 10 ug/mL; threefold dilutions
 * Also, abs panel will be slightly altered; Wayne wants to try gating with CRTH2 (CD294)
 * CD294 AlexaFluor 647
 * CD63 FITC
 * CD203c PE
 * HLA DR PE-Cy7
 * 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

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:
 * Use 50 uL cold PBS after stimulation step in order to slow/arrest degranulation.
 * Use staining buffer when making abs cocktail.
 * 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).
 * Stock IL-3 is 10 ug/mL, 5x protocol's assumed 2 ug/mL, so make sure to dilute five-fold first.
 * 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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