Samples for flow cytometric analysis are commonly prepared based on the following procedural steps:
- Prepare the sample according to cell type.
- Bone marrow (BM) and peripheral blood (PB): Prepare a blood smear, stain with Wright's stain, and scan under the microscope to identify basic cell distribution and morphology. BM can contain spicules; these samples need to be filtered.
- Tissue: Mince and filter tissues in a sterile cell culture media to release cellular components from the solid tissue form.
- Fluid: Filter fluid, prepare a cytospin, and scan under a microscope to identify cellular components.
- Obtain a white blood cell (WBC) count. Unless red blood cells are the population of interest, they should be lysed with a mild agent that will preserve the integrity of the cells targeted for analysis. If the red blood cells are not lysed, they lead to false analytic results.
- Adjust the WBC count by concentrating the WBCs to optimize for ‘staining' with monoclonal antibodies (MoAbs).
- Incubate the prepared cell concentration with assorted monoclonal antibody concentrations to allow antigen-antibody complexes to form.
- Lyse red blood cells (RBCs) with ammonium chloride or equivalent that will preserve cellular viability and integrity. The purpose is to eliminate RBCs while maintaining WBC integrity. If RBCs remain in the sample, they will interfere with the cell scatter plot and skew the results. (In some procedures, this lysis step is performed before the incubation with monoclonal antibodies)
- Centrifuge to precipitate cells.
- Pour off supernatant.
- Wash in phosphate buffered saline (PBS) or equivalent to eliminate cellular debris and unbound MoAbs, centrifuge, and decant.
- Resuspend cells in PBS.
- Analyze cells using flow cytometer.