Identify what type of cells you are analyzing, such as whole blood, PBMCs, tissue, or cell lines. Consider that rare lymphoid populations benefit from PBMC isolation, while granulocyte analysis requires whole blood.
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2
Handle cells at appropriate temperature
Keep whole blood at room temperature, but maintain PBMCs and cells in BD effects lacing solution on ice to preserve cell viability and morphology.
▶ 01:09
3
Lyse erythrocytes using appropriate buffer
Use either BD effects lacing solution (which contains fixative) or BD FarmLite buffer (without fixative) to remove red blood cells. Stain for fixative-sensitive antigens before using BD effects lacing solution.
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4
Process samples promptly or preserve them
Analyze samples immediately after collection to prevent changes in surface phenotype. If immediate processing is not possible, freeze or fix cells to preserve them for later use.
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5
Remove dead cells and debris
Filter samples through a cell strainer to remove clumping cells and dead cells. Use a viability marker to gate out dead cells and improve analysis quality and population resolution.
▶ 02:57
6
Prepare adherent cells with gentle lifting
For cultured adherent cells, use gentle lifting media such as EDTA or accutase instead of trypsin to prevent cleavage and loss of sensitive surface markers.
▶ 03:41
7
Stimulate cells under optimized conditions
Stimulate cells directly in whole blood or culture PBMCs and tissue-derived cells with appropriate medium, duration, and temperature. Note that phosphorylation assays require only minutes while cytokine assays require hours of incubation.