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:
- 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).
- Double wash cells as usual
- Incubate with DHR as usual
- Pipette cells into stim tubes here is step to use EDTA RPMI
- Stain with abs cocktail
- FACS Lyse
- Stim step was prolonged by 10 minutes (so 30 minutes total).
- 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."