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General procedure for detecting intracellular or extracellular proteins in flow cytometry.
Updated July 17, 2023.
First, we should harvest our cells or tissue and prepare a single-cell suspension. We will then transfer our single-cell suspension into 96-well plates, test tubes, or polystyrene round bottom tubes, depending on the number of cells and volumes being used.
When staining intracellular targets, we must proceed with additional fixation and permeabilization steps. Fixation is required to preserve the structure of intracellular proteins. Permeabilization disrupts the cell membrane, allowing antibodies to enter the cell and stain intracellular targets.
When staining extracellular targets, we’ll proceed immediately to the blocking step. When analyzing intracellular and extracellular targets together, we'll perform cell surface staining (see Stage 5) before fixation and permeabilization.
Useful tips for choosing suitable fixation and permeabilization methods for intracellular staining:
Materials required:
Time needed=1 hour 15 minutes approx.
Fixative | Procedure |
1-4% paraformaldehyde (PFA) | 15-20 mins on ice |
90% Methanol | 10 mins at –20°C |
100% Acetone* | 10-15 mins on ice |
Detergents | Suggested concentration |
Harsh detergents: Triton X-100, NP-40 | 0.1–1 % in PBS |
Mild detergents: Tween 20, saponin, digitonin, leucoperm | 0.2–0.5 % in PBS |
Blocking proteins and Fc domains is essential to prevent the non-specific binding of antibodies to cells.
Materials required:
Time needed= 45 min approx.
Wash cells two times with a wash buffer.
Spin cells down (200 g, 5 mins, 4°C), remove the supernatant, and resuspend the pellet after each wash.
Tip: The number of wash steps, spin time, and speed may require optimization. One wash step may suffice when using excess wash buffer and removing as much liquid as possible after centrifugation.
Proceed to antibody incubation.
We’re now ready to stain cells with fluorophore-conjugated antibodies for indirect or direct detection in the flow cytometer.
The following procedures can also be repeated and adapted for multicolor flow cytometry, in which multiple sets of fluorophore-conjugated antibodies are used against different targets. When using multiple sets of antibodies, we should minimize any overlap in the fluorophores’ emission spectra.
Materials required:
Time = 40 minutes approx.
Indirect labeling requires two incubation steps, firstly with a primary antibody, then with a compatible secondary antibody. The secondary (and not the primary) antibody has the fluorescent dye (FITC, PE, Cy5®, etc) conjugated.
Materials required:
Time =1 hour 15 minutes approx.
After antibody incubation, we can run our experiment in the flow cytometer. The procedure depends highly on the equipment used, so always refer to the manufacturer in the first instance.
For a more detailed discussion of fluorescence compensation, gating strategies, controls, and visualization methods, please refer to our flow cytometry application guide.
When surface-stained cells are live and not fixed or permeabilized, they can be separated using fluorescence-activated cell sorting (FACS). With FACS, live cells can be sorted into distinct populations based on their properties. We can then perform downstream analyses on the separated cells.
For more information, check out our free online flow cytometry training designed to help you get the best possible data from your cells.