Basophil Sensitization (11) - 8/20/10
- Had starting cell count of approx. 6.6 million PBMCs per sample (200 million total)
- Reworked fMLP and anti-IgE conceentrations:
- In past couple of months, I diluted aliquotted 4mM fMLP 4000X for concentration of 1uL, thus final 0.5uM conc. after mixing 1:1 with cells.
- Now doubled concentration, so final conc. is proper 1uM
- For preparing anti-IgE, aliquots needed to be diluted 1:9 (30uL into 270uL) for highest concentration, then diluted 1:2 twice for optimum response on curve; however, for older experiments, anti-IgE solution was mixed with cells in 3:1 ratio rather than 1:1 (need to check on this).
- Concentration used is still off, but better than before (instead of 30uL into 2670uL RPMI, I added 30uL into 1770uL RPMI).
- Running low on H12 and P4E (rDerp2-specific IgE), so used half the volume described in protocol (instead of 80uL H12 + 80uL P4E + 640 uL medium + 800 uL cells for 1600 uL total, used 40+40+320+400=800)
- Forgot to save cells for sup. control (perchloric acid treatment), so instead lysed 0.1 ng/mL rDerp2 samples.
- Checked buffy before starting experiment; was responder
- Event rate was good (15,000-20,000 basos per tube, possibly more)