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A critical step during optimization of flow cytometry experiments is reagent (antibody) titration. For optimal results you must determine the minimum amount of antibody required to achieve antigen binding saturation. This will help you improving the specificity and intensity of your fluorescent signal while minimizing background. If you are simultaneously staining with multiple colors, this titration step will also allow you to detect unexpected cross-reactivity between primary and labeled secondary antibodies.
Always remember to include negative controls of the same isotype as the labeled antibody so that you can determine the extent of background signal in your experiments. This is true when staining with a labeled primary antibody only or when using a combination of primary antibody and phycoeryhtrin labeled secondary antibody. For best results remember to always include a sample of unstained cells (incubated in parallel with your stained samples) so that you can control for background derived from auto-fluorescence. Whenever possible include cells known to express the antigen of interest as well as cells known to lack the same protein since they will help to determine the specificity of the antibodies used.
Flow Cytometry: Isotype Controls
Mouse IgG2a Isotype Control Fluorescein 010-0241
Intracellular targets entail a more challenging detection since they have additional requirements beyond antibody specificity, including the ability to successfully cross the permeable cell membrane. Optimization of cell fixation and permeabilization is a critical parameter that needs to be empirically determined in order to balance the integrity of the intracellular structures with cell permeability. Always use freshly prepared solutions of high purity paraformaldehyde for fixation and initially try mild detergents (i.e., Tween 20) for permeabilization. If your fixation and permeabilization steps need further improvement, try increasing the formaldehyde concentration gradually up to 4% or try ethanol or methanol and using other detergents including Triton X-100, or saponin without fixation or alcohol fixation.
Flow Cytometry: Intracellular Targets
Since dead cells can bind non-specifically to any antibody, it is imperative to keep those out of the analysis. The best way to do this is by using a fluorescent dye that will pass through damaged plasma membranes and thus stain dead cells. In most cases and especially when performing staining of intracellular targets, it is advisable to use dyes that covalently bind and stain non-viable cells so that they won't leak out once the cell is permeabilized.
When performing staining of cell surface markers always consider the possibility that extracellular antigens can be internalized upon antibody binding. This naturally occurring phenomenon can have a significant negative effect on the intensity of your fluorescent signal and can be prevented by:
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