Shreffler:PM Basophil Activation

Overview

Basophil activation in unfractionated samples, such as PBMC (note: basophils are less dense the Ficoll) or whole blood, can be measured by changes in the expression of cell surface markers or even intracellular events (e.g. phosphorylation, oxidative burst, calcium flux) by flow cytometry. Separate markers (e.g. CD123+ HLA-DR-) are used to specifically identify basophils in addition to those used for assessment of activation.

Materials

• RPMI medium (cellgro/Mediatech, 10-040-CV)
  • (store at 4°C in dark)
• 1 X FACS lysing solution (BD Biosciences; made from 10X stock with dH2O; store at 4°C; expires in 1 week)
• PBS + 20 mM EDTA (sterile filter, store at 4°C; expires in 1 week)
• Staining Buffer (PBS + 2 mM EDTA + 0.5% BSA) (sterile filter, store at 4°C; aliquot in hood; expires in 2 months)
• desired monoclonal antibodies

o CD63-FITC (30 μL) (BD, 557288) o CD203c-PE (30 μL)( Beckman Coulter, IM3575) o CD123 PE-Cy5 (30 μL) (BD, 551065) o CD69-APC-Cy7 (15 μL) (BD, 557756) o HLA DR-PE-CY7 (15 μL) (BD, 335795) • Stimulants o anti-IgE (Bethyl laboratories, A80-109A) o fMLP (Sigma Alderich, 47729-10MG-F)– stored 4°C