Hi there, just looking for a simple protocol stimulating 1 x 10^6 PBMCs/ mL in RMPI + 10%FBS using PHA and IL-2? I am looking for CD25, NKp44, c-kit expression and a few other markers for flow cytometry. Thanks!
Dear Chloe!
You can look at protocol stimulation of PBMC in and detecting NKp44 by FC:
Augmenting the Expression of NKp44 Molecule.pdf
c-kit (CD117) - please in link
Article CD117 (c-Kit) Is Expressed During CD8+ T Cell Priming and St...
CD25 -
Article Characterizing a soluble survival signal for activated lymph...
Flow cytometric analysis of apoptosis and cell-surface antigens
Cells were adjusted to 4×106/ml in PBS (pH 7·4 with 5% FBS). Cells (65 µl) were added to 5-ml tubes and directly fluorescenated antibodies were added into the tubes. The tubes were incubated for 30 min at 4°, washed once with PBS (pH 7·4) and reconstituted in PBS (pH 7·4). The cells were analysed using flow cytometry (three colors). Antibodies used included: CD3-phycoerythrin-cyanin 5.1 (PC5), T4-fluorescein isothiocyanate (FITC), CD4-phycoerythrin, red dye 1 (RD1), T8-FITC, CD8-RD1, B4-RD1 (CD19), B4-FITC (CD19), CD69-phycoerythrin (PE), CD25-PC5, CD122-PE, CD132-PE, CD95-PE and CD14-PE (purchased from Beckman Coulter, Miami, FL). For apoptosis, the Annexin V-FITC kit was used (Immunotech, Beckman Coulter) to stain the cells. Briefly, 4×105 cells were washed once with PBS and adjusted to a concentration of 4×106/ml with 100 µl of buffer; 1 µl of Annexin V and 5 µl PI (propidium iodide) were added and mixed. The cells were then incubated at 4° for 10 min in the dark and analysed by flow cytometry without washing.
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