Shreffler:PM Basophil Activation: Difference between revisions
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| Line 6: | Line 6: | ||
**Store at 4°C in dark | **Store at 4°C in dark | ||
*1 X FACS lysing solution (BD Biosciences) | *1 X FACS lysing solution (BD Biosciences) | ||
**Make from 10X stock with dH2O; store at 4°C; expires in 1 week | |||
*PBS + 20 mM EDTA | *PBS + 20 mM EDTA | ||
**Sterile filter, store at 4°C; expires in 1 week | **Sterile filter, store at 4°C; expires in 1 week | ||
| Line 12: | Line 12: | ||
**Sterile filter, store at 4°C; aliquot in hood; expires in 2 months | **Sterile filter, store at 4°C; aliquot in hood; expires in 2 months | ||
*Desired monoclonal antibodies | *Desired monoclonal antibodies | ||
**CD63-FITC (30 μL, BD 557288) | |||
**CD203c-PE (30 μL, Beckman Coulter IM3575) | |||
**CD123 PE-Cy5 (30 μL, BD 551065) | |||
**CD69-APC-Cy7 (15 μL, BD 557756) | |||
**HLA DR-PE-CY7 (15 μL, BD 335795) | |||
*Stimulants | |||
**Anti-IgE (Bethyl laboratories A80-109A) | |||
**fMLP (Sigma Alderich 47729-10MG-F)– stored at 4°C | |||
Revision as of 08:42, 21 December 2009
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)
- Make 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
- CD63-FITC (30 μL, BD 557288)
- CD203c-PE (30 μL, Beckman Coulter IM3575)
- CD123 PE-Cy5 (30 μL, BD 551065)
- CD69-APC-Cy7 (15 μL, BD 557756)
- HLA DR-PE-CY7 (15 μL, BD 335795)
- Stimulants
- Anti-IgE (Bethyl laboratories A80-109A)
- fMLP (Sigma Alderich 47729-10MG-F)– stored at 4°C
