Immunophenotyping by Flow Cytometry


What is Immunophenotyping?

Immunophenotyping is the characterization and classification of cells in a heterogeneous mixture of cells such as blood, synovial fluid, other aqueous liquids, or solid tissues. It uses the expression of specific genes and combinations of genes to categorize cell types based on known functional characteristics.

Although flow cytometry can be used for a wide variety of applications, immunophenotyping is probably the most common. It is possible to perform immunophenotyping using other techniques like microscopy or single cell RNAseq but flow cytometry is by far the most widely used method due to its ability to generate highly detailed information quickly.

Immunophenotyping is invaluable for researchers seeking to understand the immune system. It is used extensively in the fields of immunology, cancer research, stem cell biology, rheumatology, reproductive, and developmental biology. It also has many clinical applications including cancer diagnosis and monitoring, immunological monitoring of HIV, and diagnosis of immunological dysfunction.

Briefly, in flow cytometry-based immunophenotyping, a heterogeneous, suspension of cells is incubated with antibodies that recognize specific proteins present, usually on the surface of cells. The antibodies used are either directly or indirectly linked to fluorescent dyes. This allows cells expressing specific protein markers to be detected based on their fluorescence.

The fundamental advantage of using flow cytometry over other techniques for immunophenotyping is its ability to provide complex data at speed and at a single cell level. Bio-Rad developed the ZE5 Cell Analyzer with the aim of providing high parameter data faster than ever before.

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The Perfect Balance of High Parameter Data & Speed with Reproducibility

High Parameter Data

With up to 5 lasers and 30 parameters, the ZE5 Cell Analyzer can perform immunophenotyping in depth. In this example, we show a 27-color immunophenotyping panel designed using our cutting edge StarBright™ Dyes to characterize the major human leukocyte sub populations. You can also download the full PDF poster and a technical note with methods and additional data.

27-Color Immunophenotyping Panel Including StarBright, UltraViolet, Violet , Blue, Yellow, and Red Dyes

Target StarBright Range Fluorescent Dye Product Code
HLA DP DQ DR UltraViolet SBUV400 MCA477SBUV400
CD20 UltraViolet SBUV510 MCA1710SBUV510
CD33 UltraViolet SBUV575 MCA1271SBUV575
CD163 UltraViolet SBUV665 Coming soon
CD28 UltraViolet SBUV740 MCA709SBUV740
CD62L UltraViolet SBUV795 MCA1076SBUV795
CD56 N/A BV421 N/A
CD24 Violet SBV440 Coming soon
CD45RA Violet SBV515 MCA88SBV515
CD45RO Violet SBV610 MCA461SBV610
CD40 Violet SBV670 Coming soon
CD2 Violet SBV710 MCA1194SBV710





CD3 Blue SBB580 MCA463SBB580
CD11b Blue SBB700 Coming soon
CD10 Yellow SBY575 MCA1556SBY575
CD4 Yellow SBY605 MCA1267SBY605
CD45 Yellow SBY665 MCA87SBY665
CD27 Yellow SBY720 MCA755SBY720
CD38 Yellow SBY800 MCA1019SBY800
CD16 N/A A647 MCA5665A647
CD31 N/A A700 MCA1738A700
CD19 Red SBR775 Coming soon
CD8 Red SBR815 Coming soon
PI N/A L/D 1351101

Initial Plots

27-Color Multiplex Panel

27-Color Multiplex Panel. Red blood cell lysed human peripheral blood was stained with a live/dead dye (propidium iodide, (PI)) and a 26-color antibody panel in FACS Buffer allowing identification of multiple cell lineages and subsets.

Full Gating Strategy

Click each image to see the full gating strategy:


Speed with Reproducibility

The ZE5 Cell Analyzer can acquire complex immunophenotyping data at rates that significantly exceed conventional instruments. Its innovative fluidics and electronics synergize to enable it to process a 96-well plate in less than 15 minutes whilst still being able to collect tens of thousands of events per well. This means that even complex assays such as the one detailed above can be performed at the speed of a screening instrument and without compromising reproducibility.

To demonstrate this, we repeated the 27-color panel shown above using high throughput mode for sample acquisition. The data shows a high level of reproducibility between high throughput runs and is consistent with standard mode runs. Whilst data quality was preserved the run time for each well was reduced from 3 minutes 21 seconds in standard mode, to 8 seconds in high throughput mode.

Example 27-Color Panel Comparison of Standard and High Throughput Data Acquisition

Chart of 27-Color Panel Comparison

This example shows six replicates of the 27-color panel data shown previously, a single sample was collected in high throughput (n=4) and standard mode (n=2). Each standard mode replicate was collected in 3 minutes and 21 seconds, while each high throughput mode replicate was collected in 8 seconds.

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    Unlock the Power of High Speed Immunophenotyping

    Contact a flow cytometry specialist to discuss how the ZE5 Cell Analyzer can deliver a significant increase in the quantity and quality of your immunophenotyping data.

Powerful Instruments with Cutting Edge Dyes

For the ultimate synergy of instrument and reagents combine the speed and power of the ZE5 Cell Analyzer with our range of innovative StarBright Dyes.

Additional Resources

Explore other example immunophenotyping panels analyzed using the ZE5 Cell Analyzer and see how StarBright Dyes can enhance any panel on any instrument. Includes detailed immunophenotyping protocols.

StarBright Dyes: Build Bigger, Better Panels With Superior Dyes Posters

Flow Immunophenotyping Tools & Services

Building new immunophenotyping panels can be challenging. We have developed a range of resources that can help you get the most out of your flow cytometer regardless of manufacturer or configuration.

Further Reading

Quantification of T cell Subsets in Stem Cell Transplant Patients Using a 21-Color Panel

A 19-Color Panel for the Immunophenotyping of Normal and Malignant Leukocytes by Flow Cytometry