5 multicolour flow cytometry reagents for immunophenotyping

Dual CD4 CD3 leukocyte staining by FCM Exbio tebu-bio

Multicolour fluorochrome reagents have spurred researchers’ capacity to analyze cell populations and to isolate cell subsets by flow cytometry. In this post, let’s take at look at the advantages of 5 optimized combinations of mixed fluorochromes you should consider for your next immunophenotyping assays by multicolour flow cytometry.

Multicolor FCM CD3 FITC CD PE CD45 PerCP CD APC ED7045 Exbio tebu-bio

Multicolor staining by FCM with CD3 FITC / CD PE / CD45 PerCP / CD APC cat. nr ED7045. Souce: Exbio / tebu-bio.

KOMBITEST™ Assay kits are cost-effective premixed combinations of mouse monoclonal antibodies to detect specific human antigens of interest (a.k.a. biomarkers) expressed on the surface of leukocytes by Flow Cytometry (FCM). These monoclonal antibodies are conjugated with different well-selected fluorochromes. When excited via laser beam from a flow cytometer, subsequent light emissions of each individual fluorochromes are collected and analyzed by a flow cytometer. These FCM Assay Kits are compatible with the specific staining of blood cells by incubating blood samples with the KOMBITEST™ reagents followed by a lysis of red blood cells to allow the analysis of leukocytes. The fluorescence intensity difference for each fluorochrome, enables the separation of cell subsets based on the expression of analyzed antigens.

Below, I have selected 5 ready-to-use multi-color KOMBITEST™ combinations for their stabilty and ease of use when running your immunophenotyping  assays while ensuring biologically relevant results.

Combination#1: 2-color for Human CD4 / CD8 analysis

The KOMBITEST™ CD4 (clone MEM-241) FITC / CD8 (clone MEM-31)  PE is designed for percentage enumeration of mature human helper/inducer (CD4+) and suppressor/cytotoxic (CD8+) lymphocytes in erythrocyte-lysed whole blood enabling the assessment of the helper/suppressor ratio (CD4+ / CD8+).

To know more: KOMBITEST™ CD4 FITC / CD8 PE (cat. nr ED7052-T050 – 50 tests).

Combination#2: 3-color for Human CD4 / CD8 / CD3 analysis

The KOMBITEST™ CD4 (clone MEM- 241) FITC / CD8 (clone MEM-31) PE / CD3 (clone UCHT1) PE-Cy™5 is designed for percentage enumeration of mature human helper/inducer (CD3+CD4+) and suppressor/cytotoxic (CD3+CD8+) T lymphocytes in erythrocyte-lysed whole blood.

To know more: KOMBITEST™ CD4 FITC / CD8 PE / CD3 PE-Cy™5 (cat. nr ED7058-T050 – 50 tests).

Combination#3: 4-color for Human CD3 / CD8 / CD45 / CD4 analysis

The KOMBITEST™ CD3 (clone UCHT1) FITC / CD8 (clone MEM-31) PE / CD45 (clone MEM-28) PerCP / CD4 (clone MEM-241) APC is designed for percentage enumeration of mature human helper/inducer (CD3+CD4+) and suppressor/cytotoxic (CD3+CD8+) T lymphocytes in erythrocyte-lysed whole blood.

To know more: KOMBITEST™ CD3 FITC / CD8 PE / CD45 PerCP / CD4 APC (cat. nr ED7045-T050 – 50 tests).

Combination#4: 4-color for Human CD3 / CD16+CD56 / CD45 / CD19 analysis

The KOMBITEST™ CD3 (clone UCHT1) FITC / CD16+CD56 (clones 3G8 & LT56) PE / CD45 (clone MEM-28) PerCP / CD19 (clone LT19) APC is designed for identification and enumeration of human T, natural killer, and B lymphocytes in erythrocyte-lysed whole blood using Flow Cytometry.

To know more: KOMBITEST™ CD3 FITC / CD16+CD56 PE / CD45 PerCP / CD19 APC (cat. nr ED7074-T050 – 50 tests).

Combination#5: 6-color for Human CD3 / CD4  / CD8  / CD16+CD56 / CD19 / CD45 analysis

The KOMBITEST™ CD3 (clone UCHT1) FITC / CD4 (clone MEM-241) PE-Cy™7 / CD8 (clone MEM-31) APC-Cy™7 / CD16+CD56 (clones 3G8 & LT56) PE / CD19 (clone LT19) APC / CD45 (clone MEM-28) PerCP-Cy™5.5 is designed for identification and enumeration of mature human T, B, and NK lymphocytes as well as helper/inducer (CD3+CD4+) and suppressor/cytotoxic (CD3+CD8+) T lymphocyte subsets in erythrocyte-lyzed whole blood using Flow Cytometry.

To know more: KOMBITEST™ CD3 FITC / CD4 PE-Cy™7 / CD8 APC-Cy™7 / CD16+CD56 PE / CD19 APC / CD45 PerCP-Cy™5.5 (cat. nr ED7075-T050 – 50 tests).

Which red cell lysis buffers?

Various sources of ready-to-use buffers for red blood cell lysis are commercially available. Here is a selection of cost-effective alternatives compatible with single or multi-color FCM for both lyse/wash and lyse/no wash protocol.

  • EXCELLYSE® Easy (10x concentrated lysing solution).
  • EXCELLYSE® Live (10x concentrated lysing solution) for use when viable leukocytes are required after red blood cell lysis (compatible with both lyse/wash and lyse/no wash protocols).
  • EXCELLYSE® I (red blood cell lysing solution following antibody staining of human peripheral blood leukocytes – optimized for use with BD Biosciences flow cytometers.

What about you? Any questions or comments? What are your preferred combinations of multiple flurochromes for FCM analysis?

Please feel free to contact me with the form below!

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Philippe Fixe, PhD
Written by Philippe Fixe, PhD
Philippe Fixe is Marketing Director at tebu-bio, passionate about innovation and R&D in Life sciences, Biotechnology, Medical research, Drug discovery, and also a keen photographer.