Flow cytometry protocol

General procedure for detecting intracellular or extracellular proteins in flow cytometry.

Stage 1 - Sample preparation

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.

Materials required

Steps

20 minutes approx

Harvest and wash cells according to the manufacturer's guidance.

Prevent cell damage by avoiding bubbles, vigorous vortexing, aspirating the entire solution during buffer exchange, and excessive centrifugation.
A red blood cell lysis buffer will lyse red blood cells, which might interfere with the analysis of leukocytes (nucleated cells).

Determine the total cell number and check cell viability.

Spin down and resuspend cell samples in an ice-cold suspension buffer.

Higher cell concentrations might clog up the flow cytometer system and affect resolution.
Spin times and speeds may require optimization. In general, cells should be centrifuged sufficiently so the supernatant fluid can be removed with little loss of cells but not so hard that the cells are difficult to resuspend.

Proceed to stain with a viability dye.

Stage 2 - Live/dead staining with a viability dye

As dead cells are prone to bind to antibodies non-specifically, we must exclude those cells from the analysis. Using viability dyes allows us to distinguish between live and dead cells and exclude the dead ones during data acquisition and analysis.

DNA binding dyes, such as 7-AAD, DAPI, and TOPRO3, are often used as viability dyes for live/dead staining, as they cannot penetrate the cell membrane of live cells. The compromised cell membrane found in dead cells allows these dyes access to DNA, to which they bind and emit fluorescence.

However, these dyes cannot be used for live/dead staining with fixed cells, where cell membranes would be compromised in all cells. In this case, we must use amine-reactive fixable cell viability dyes.

Materials required

Steps

Stain cells with a viability dye.

Keep fluorophores in the dark to avoid photobleaching.
Choose a dye with an emission spectrum that does not overlap with the fluorophores used for immunostaining.

Wash cells two times with wash buffer.

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 blocking when detecting extracellular targets or to fixation and permeabilization for intracellular targets.

Stage 3 - Fixation and permeabilization (optional - only for intracellular staining)

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 intra and extracellular targets together, we'll perform cell surface staining (ie, Stage 5) before fixation.

Helpful tips for choosing suitable fixation and permeabilization methods for intracellular staining:

Materials required

Steps

1 hour 15 minutes approx

Fix the cells in your chosen fixative.

Fixative
1-4% paraformaldehyde (PFA)
90% methanol  
100% acetone
Procedure
15-20 min on ice
10 min at -20°C
10-15 min on ice
Polystyrene/plastic tubes are not suitable for use with acetone.

The fixation will require optimization for different antigens.

Some epitopes are very sensitive to methanol, so try acetone instead if any issues are occurring with detection.

Wash the cells two times with suspension buffer.

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.

Permeabilize cells by incubating them with a suitable detergent.

Detergents
Harsh detergents: Triton X-100, NP-40, Column1
0.1-1% in PBS
Mild detergents: Tween 20, saponin, digitonin, leucoperm
0.2-0.5% in PBS
This step is not required if acetone has been used as a fixative, as acetone also permeabilizes cells.

The optimal detergent will depend on the protein and its localization. Harsh detergents, such as Triton or NP-40, partially dissolve the nuclear membrane and are, therefore, suitable for nuclear antigen staining. In contrast, mild detergents, such as Tween 20 or saponin, enable antibodies to go through pores without dissolving the plasma membrane, which makes them suitable for antigens in cytoplasm, or cytoplasmic face of the plasma membrane and soluble nuclear antigens.

The concentration of detergent should be optimized for the samples being used.

Permeabilization will affect the light scatter profiles of the cells on the flow cytometer; keep that in mind when gating on cell populations during the detection and data analysis (Stage 6).

Wash the cells two times with the suspension buffer.

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.

Stage 4 - Blocking

Blocking proteins and Fc domains is essential to prevent the non-specific binding of antibodies to cells.

Materials required

Steps

45 minutes approx

Block Fc receptors with a blocking buffer.

Blocking buffers:

Wash cells two times with the wash buffer.

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.

Stage 5 - 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. We should minimize any overlap in the fluorophores’ emission spectra when using multiple sets of antibodies.

Materials required

Steps

40 minutes approx

Dilute the conjugated primary antibody in the suspension buffer.

Titrating the antibody by performing serial dilutions will help find the antibody concentration that works best for your experiment.

Incubate cells in the pre-diluted primary antibody.

Keep fluorophores in the dark to avoid photobleaching.    

Fixed cells can be incubated at room temperature or 4°C.

This step may require optimization.

Wash the cells two times with the suspension buffer.

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 detection in the flow cytometer as soon as possible.

Do not fix cells if you intend to study them live.

The best results are obtained immediately after incubation.

Keep the cells in the dark on ice or at 4°C in a fridge until your scheduled analysis time.

Note that fixation won't be compatible with non-fixable cell viability dyes (if previously used).

Stage 6 - Detection and data analysis

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.