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Though dye fluorescence is most commonly assayed in flow cytometry, valuable information can be determined from the autofluorescence levels of unstained cells. Cells are naturally autofluorescent, especially at lower wavelengths of the visible spectrum, and the scattered light can provide information on the cell size, granularity, surface topography, and nuclear: cytoplasmic ratio. For instance, an analysis of white blood cells allows identification of different leukocyte types.
However, the vast majority of flow cytometry experiments involve the use of fluorochromes. These are small organic molecules or proteins such as allophycocyanin (APC) with fluorescent properties. Fluorochromes are often conjugated to primary or secondary antibodies to mark targets or biomarkers. An alternative to antibody staining is to use cells expressing, either stably or transiently, proteins of interest fused with fluorescent proteins such as GFP, Venus, or mRFP.
The choice of the fluorochrome depends on the experimental setup:
To minimize spectral overlap, avoid using fluorochromes with similar excitation spectra if they are excited by the same laser.
Some fluorochromes add significant molecular weight to the labeling reagents – this may reduce their ability to permeate the cell membrane to stain intracellular targets.
Be sure to properly handle tandem dyes – avoid freeze-thaw cycles and exposure to light to minimize decoupling.
If fluorophore reagents are used, cells are stained and then washed prior to analysis to remove the excess of unbound dyes. Table 1 shows tips on staining procedures with different fluorophore reagents. Permeabilization is required for the analysis of intracellular proteins using antibodies.
Table 1. Types of fluorophore reagents commonly used in flow cytometry.
|Reagent||Example||Live cells||Fixed cells||Permeabilization|
|Viability dyes||Propidium iodide||yes||no||no|
|Apoptotic markers||Fluorescently labeled annexin V||yes||no||no|
|Fluorescent proteins expressed by cells||GFP-LC3||yes||yes||Not required|
|Fluorescently labeled antibodies against extracellular proteins||E-cadherin (see Figure 2)||yes||yes||Not required|
|Fluorescently labeled antibodies against intracellular proteins||Lamin A/C (see Figure 2)||Technically challenging||yes||Required|
Figure 2. Flow cytometry is suitable for analyzing both extracellular and intracellular targets.
Left: 1X10^6 HepG2 cells were stained with 2ug E-cadherin antibody (20874-1-AP, red) and control antibody (blue). Fixed with 90% MeOH blocked with 3% BSA (30 min). Alexa Fluor™ 488-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) with dilution 1:1000.
Right: 1X10^6 HEK-293T cells were stained with 0.2ug Lamin A/C antibody (10298-1-AP, red) and control antibody (blue). Fixed with 90% MeOH blocked with 3% BSA (30 min). Alexa Fluor™ 488-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) with dilution 1:1000.