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
- Cell suspension
- Polystyrene round bottom 12 x 75 mm2 Falcon tubes/96-well plate (or any other container compatible with your centrifuge)
- Suspension/washing buffer (PBS, 5-10% fetal calf serum (FCS))
- Optional - red blood cell lysis buffer (for example ab204733)
Steps
Harvest and wash cells according to the manufacturer's guidance.
- For blood samples, we suggest incubating samples with RBC lysis buffer before proceeding.
Determine the total cell number and check cell viability.
- In general, viability should be 90-95%.
Spin down and resuspend cell samples in an ice-cold suspension buffer.
- Centrifuge at ~200 g for 5 min at 4°C.
- Recommended cell concentration for suspension: 0.5–1 x 106 cells/mL.
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.
- Incubate cells with dye in the dark at 4°C, according to the manufacturer's instructions.
Wash cells two times with wash buffer.
- Spin cells down (200 g, 5 min, 4°C), remove the supernatant and resuspend the pellet after each wash.
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:
- Antigens close to the plasma membrane and soluble cytoplasmic antigens will require mild cell permeabilization without fixation.
- Cytoskeletal, viral, and some enzyme antigens usually give optimal results when fixed with a high concentration of acetone, alcohol, or formaldehyde.
- Antigens in cytoplasmic organelles and granules will require a fixation and permeabilization method, depending on the antigen.
- The epitope needs to remain accessible.
Materials required
- Cell suspension
- Suspension buffer (PBS, 5-10% FCS)
- Fixative (for example, 1-4% paraformaldehyde, 90% methanol, or acetone)
- Permeabilization solution (for example, Triton X-100, NP-40, or Saponin)
- Alternatively, you can also use our fixation and permeabilization kit for flow cytometry (ab185917), which is suitable for most sample types.
Steps
Fix the cells in your chosen fixative.
- Spin down cells to a pellet (200 g, 5 min, 4°C), discard the supernatant, and resuspend the pellet in fixative.
- Incubate cells with the fixative as indicated below.
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.
- Spin down cells to a pellet (200 g, 5 min, 4°C), discard the supernatant, and resuspend in wash buffer.
Permeabilize cells by incubating them with a suitable detergent.
- Spin down cells to a pellet (200 g, 5 min, 4°C), discard the supernatant, and resuspend the pellet in a detergent solution.
- Incubate cells in the detergent for 10-15 mins at room temperature.
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.
- Spin down cells to a pellet (200 g, 5 min, 4°C), discard the supernatant, and resuspend the pellet in the wash buffer.
Stage 4 - Blocking
Blocking proteins and Fc domains is essential to prevent the non-specific binding of antibodies to cells.
Materials required
- The choice of materials will depend on the type of cells analyzed and, if applicable, the secondary antibody used.
- FcR Blocking buffer (for example, 2-10% goat serum, human IgG, or mouse anti-CD16/CD32)
- Suspension buffer (PBS, 5-10% FCS) 5-10%
Steps
Block Fc receptors with a blocking buffer.
- Spin down cells to a pellet (200 g, 5 min, 4°C), discard the supernatant, and resuspend the pellet in the blocking buffer.
- Incubate cells with one of the buffers below for 30-60 mins in the dark at 4°C.
Blocking buffers:
- 2-10% goat serum
- Human IgG
- Mouse anti-CD16/CD32
Wash cells two times with the wash buffer.
- Spin cells down (200 g, 5 min, 4°C), remove the supernatant and resuspend the pellet after each wash.
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
- Conjugated primary antibody
- Samples (cell suspension at 0.5 - 1 x 106 cells/mL)
- Suspension buffer (PBS, 5-10% FCS)
Steps
Dilute the conjugated primary antibody in the suspension buffer.
- Suggested dilutions for each antibody will often be provided on the datasheet.
Incubate cells in the pre-diluted primary antibody.
- Spin down cells to a pellet (200 g, 5 min, 4°C), discard the supernatant, and resuspend the cells in the primary antibody solution.
- Incubate in the dark for 20-30 min at 4°C.
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.
- Spin cells down (200 g, 5 min, 4°C), remove the supernatant, and resuspend the pellet after each wash.
Proceed to detection in the flow cytometer as soon as possible.
- If the cells are not analyzed in the flow cytometer immediately after antibody staining (within 1 hour) and were not stained earlier, stained cells can be fixed at this step (1-4% PFA, 20 min, 4°C). Fixation helps preserve the cells for several days, stabilizing the light scatter and inactivating most biohazardous agents. The controls will require fixation using the same procedure. Note that fixation will kill cells.
- After fixation, wash cells three times and store cell suspension in the suspension buffer.
- Follow the manufacturer's instructions.
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.