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Every flow cytometry (or FACS) experiment begins with sample preparation. Check out our flow cytometry sample preparation guide to learn how to prepare your samples for flow cytometric analysis.
This information is to serve as a guide as individual investigators may need to optimize protocols for their particular cell type.
Cells for flow cytometry analysis are usually derived from four main sources:
Regardless of the source, the final cell preparation should be:
a homogenous single-cell suspension free of clumps and dead cell debris at a density of 106-107 cell per ml suspended in a suitable staining buffer.
PBMCs isolated from whole blood through Ficoll gradient centrifugation or RBC lysed whole blood or non-adherent cultured cells are readily available for flow cytometric analysis. Adherent cultured cells or cells present in the solid organs should be first made into a single cell suspension before flow analysis by using enzymatic digestion or mechanical dissociation of the tissue, respectively. Mechanical filtration should be followed to avoid unwanted instrument clogs or lower quality flow data. Use the following sample preparation protocols based on your appropriate starting materials.
Key Reagents – PBS, staining buffer
Key Reagents – PBS, staining buffer, 0.25% trypsin
Key reagents – PBS, staining buffer, culture medium with 10% FBS
Key reagents – PBS, staining buffers, suitable gradients medium like Ficoll or Histopaque
Key reagents – PBS, staining buffer (see appendix), RBC lysis buffer [check Appendix for recipe or use a commercially available buffer]
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