Flow Cytometry Protocol | 10 Hints & Tips

Flow Cytometry Protocol

The meaning is in the name:

Flow = in motion, Cyto = cells, Metry = measure.

What is Flow Cytometry?

Flow cytometry measures the properties of cells while in a fluid stream. It enables single cell analysis of complex cellular systems (e.g. blood) very rapidly (100s of cells/second) it also allows you to look at various cellular properties such as size, granularity, fluorescence intensity per cell.

Flow Cytometry can be used for cell counting, cell sorting, biomarker detection and protein engineering. Cell components are fluorescently labelled and then excited by the laser to emit light at varying wavelengths. Flow cytometry results enable you to pinpoint exactly which cells are activated or if there are multiple pathways being activated.

An example of 2 parameters you can select in flow cytometry are forward scatter (FSC) and side scatter channel (SSC). The intensity of FSC correlates with particle size and can be used to distinguish cellular debris from living cells. SSC gives information about the granular content of a particle. The FSC and SSC are unique for every particle and can used to distinguish different cell types.

Flow Cytometry Protocols

Below you will find 2 sample flow cytometry protocols in addition to some hints and tips for flow cytometry experiments.

Flow Cytometry Protocol 1

This protocol for flow cytometry sample fixation is a quick protocol to prepare cells for cell cycle analysis and DNA labelling. This protocol will help you measure the phase of the cell cycle, G1, S and G2/M when stained with propidium iodide.

Solutions and Reagents

  • Pipettes
  • Tips
  • Ethanol
  • RNAse
  • PBS
  • Propidium iodine
  • And of course a flow cytometer.

Flow Cytometry Sample Fixation

  1. Harvest K562 cells from treated samples by centrifugation at 270 x g for 5 min to pellet the cells
  2. Carefully aspirate the supernantant without disturbing the pellet.
  3. Wash the pellet in ice-cold phosphate buffered saline (PBS)
  4. Centrifuge at 270 x g for 5 min.
  5. Resuspend the cells in 200 µl of PBS
  6. Add 2 ml of ice-cold ethanol (70% (v/v)) before incubating at 4 °C overnight prior to sample analysis.

Fluorescent DNA staining

  1. Centrifuge fixed samples at 270 x g for 5 min.
  2. Aspirate the Ethanol and resuspend in 500 µl of PBS
  3. Add RNase A (30mg/ ml) and propidium iodide (300 µM) and mix.
  4. Incubate samples in the dark at 37 °C for 30 min prior to analysis.
  5. Cell cycle profile was analysed using the COULTER© EPICS© XL-MCL Flow Cytometer.

Flow Cytometry Protocol 2

This protocol Flow cytometry was used to determine the effects of a particular treatment on cell surface expression of receptors, such as CXCR4.

Solutions and Reagents

  • Pipettes
  • Tips
  • RPMI + 0.5% BSA.
  • 100 ng/ ml SDF-1α
  • Phycoerythirin (PE)-labelled antibodies
  • Ethanol
  • RNAse
  • PBS
  • A flow cytometer.

Flow Cytometry Sample Fixation

  1. Seed Jurkat T cells at 5 x105 cells/ ml and serum starve for 2 hours prior to experiment in serum free RPMI + 0.5% BSA.
  2. Stimulate cells with 100 ng/ ml SDF-1α under discontinuous stirring conditions
  3. Stop the activation by centrifugation for 30 sec at 850 xg on a benchtop centrifuge
  4. Wash cells with ice-cold PBS
  5. Stain with Phycoerythirin (PE)-labelled antibodies against cell surface receptors in 1% BSA/ PBS and incubate for 30 min on ice.
  6. Wash twice with 1% BSA/ PBS
  7. Fix in 1% PFA on ice
  8. Store for up to 24 hours at 4°C or analyse immediately by flow cytommetry.

Fluorescent DNA staining

  1. Cell surface expression of receptors, such as CXCR4 was measured with the DAKO CyAn ADP (Advanced Digital Processing)
  2. The results were analysed using associated the Dako Summit (version 4.3) software to generate histograms. The data then underwent statistical analysis and was graphed using Graph Pad Prism.

Flow Cytometry Hints & Tips

1. Sample Preparation

This is the start of your experiment and the most important part. Ensure sample preparation is fully optimized, whether you are using adherent or tissue derived cells. If the cells are not harvested correctly this can lead to reduced cell viability. Furthermore the use of any enzymatic procedures should not have any effect on the expression of the markers that are being studied.

2. Fixation and permeabilization reagents

Not all fixation and permeabilization solutions work for all experimental settings and can vary depending on the antigen being examined.

3. Your choice of Fluorochrome

Before choosing your fluorochrome it is essential that you know the configurations of laser, filters and the emission spectra characteristics of the fluorochrome that you are considering. You must also know the capabilities of the instruemnt you will be using. No two cytometers are alike in their capabilities. If you have a flow cytometry core with knowledgeable staff, be sure to ask them what their favourite 4, or 5, or 6-colour panel is. They should also be able to tell you what the limitations of certain colours on a given instrument may be. Always be sure to select a bright fluorochrome to detect antigens that are expressed at low density, and dim fluorochromes to detect antigens that are expressed at high density. The choice of fluorochrome is also important for compensation, and the possibility that ‘bright’ fluorochromes might cause problems with spectral overlap should be considered.

4. Selecting the right controls

As with all experiments the choice of control is critical as it ultimately allows you to determine whether or not you assay is working correctly and forms an essential part of your results. It is recommend that the following types of controls to be incorporated into your workflow:

    1. Set-up or instrument controls, used to correctly adjust instrument voltage gains and compensation
    2. Gating controls, used to distinguish specific from non-specific binding
    3. Experimental controls to provide biologically meaningful information

The table below lists the appropriate controls relevant to each of the three categories above:

Control Use

Unstained Cells

Negative control to determine background & autofluorescence, Setting of PTM Voltages

Fully Stained Cells

Check for any off-scale events, Setting PTM voltages

Cell samples/beads stained with single fluorescent markers

Compensation, Gating – eliminating non-specific staining from analysis

Isotype control

Gating – eliminating non-specific staining from analysis

Fluorescence Minus One (FMO) staining

Gating – reduce fluorescence spillover

Unstimulated or untreated sample

Experimental negative control

Positive cells (stimulated)

Experimental positive control

Cells stained with a viability dye, e.g., propidium iodide

Discriminating viable from non-viable cells

5. Identify dead cells

Identification and elimination of dead cells if possible can help reduce no specific staining. Dead cells can be detected using a DNA dye which penetrates cells with a loss of membrane integrity.

6. Identification and gating out of doublets

Doublets occur when two cells are stuck together and are analyzed as one. This will falsely increase the fluorescence intensity of the ‘cells’ passing through the laser interrogation point. Gate out doublets by by creating a window using the Forward Scatter INT versus Forward Scatter Time-of-Flight (ToF) or Side Scatter INT versus Side Scatter Time-of-Flight (ToF). Following on from this, filter the cells before analyzing them in order to exclude cell clumps.

7. Sample Storage

In an ideal world samples should be analysed immediately however this doesn’t always happen. If you cant analyse your samples straight away resuspend in 100µl fixative (e.g. 1-2% formaldehyde) and store at 4°C, protected from the light for up to 24 hours.

8. Reduce fluorescent signal noise

A critical step during optimization of flow cytometry experiments is reagent (antibody) titration. This will help you improve the specificity and intensity of your fluorescent signal while minimizing background.

9. Reduce fluorescent signal noise

A critical step during optimization of flow cytometry experiments is reagent (antibody) titration. This will help you improve the specificity and intensity of your fluorescent signal while minimizing background.

10. Always protect from light degradation

To prevent fluorochrome degradation by ambient light, plates or tubes containing cells that have been stained or are in the process of being stained should be protected from by covering samples with foil or another light-blocking mechanism

Written by Sean Mac Fhearraigh

Seán Mac Fhearraigh PhD is a co-founder of Assay Genie. Seán carried out his undergraduate degree in Genetics at Trinity College Dublin, followed by a PhD at University College Dublin. He carried out a post-doc at the Department of Genetics, University of Cambridge. Seán is now Chief Technical Officer at Assay Genie.

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7th Nov 2022 Sean Mac Fhearraigh

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