(Difference between revisions)
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|-|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. as CD123+ HLA-DR-) are used to specifically identify basophils in addition to those used for assessment of activation. |+|
in in the cell intracellular . used to .
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RPMI medium (cellgro, 10-040-CV; store at 4°C in dark) |+|
|-|* 1 X FACS lysing solution (made from 10X stock with dH<sub>2</sub>O; store at 4°C; expires in 1 week) |+|
+ 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 (e. g. CD63-FITC, CD203c-PE, CD123 PE-Cy5, CD69-APC-Cy7, HLA DR-PE-CY7, CD3-APC, CD14-APC, CD19-APC, CD41a-APC) |+|
|-|'''May use other antigens for specific application. This will give an idea of the typical dose range we use for antigen- specific responses and controls. ''' |+|
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CFSE is used to fluorescently label live cells in order to track them. 5,6-CFDA/SE is membrane permeable, but trapped in the cell when converted by intracellular esterases. CFSE is equally partitioned to daughter cells during division and can be used to measure cell proliferation.
- 15 mL polypropylene tubes
- 5,6-CFDA/SE Invitrogen C1157 dissolved in DMSO at 5 mM and stored at -20°C.
- Suspend PBMCs at 10x106 cells/mL in PBS alone.
- Ensure that cells are uniformly suspended when CFSE is added.
- If CFSE negative control is needed, remove cells now.
- Make '2X' concentration (10 μM) in PBS
- For example: add 5 mL PBS + 10 μL 5 mM CFSE
- Combine 1:1 PBMC cells + CFSE (e.g. 5 mL PBMCs + 5 mL CFSE) in 15mL tube and mix completely.
- Place in 37°C H2O bath x 10 min.
- Wash in 10 mL complete medium.
- Resuspend in medium at desired density
discuss this protocol
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