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Basic T cell staining (4c method)


This protocol describes phenotyping of T cells in a whole blood assay by 4 colour flow cytometry.

Required patient material

1ml EDTA-anti-coagulated or heparinized blood

Required reagents

FACSFlow (BD Biosciences)

Optilyse B (Beckman-Coulter)

Deionised water

* reagents of other manufacturer’s may be used after testing


4 colour flow cytometer as for example FACSCalibur with 488nm-laser and 635nm-diode-laser (BD Biosciences)

* equipment of other manufacturer’s may be used


Antibody Clone Fluorchrome Order No. Provider
CD3 SK7 PerCP 345766 BD
CD4 SK3 APC 345771 BD
CD4 SK3 PerCP 345770 BD
CD8 SK1 APC 345775 BD
CD27 M-T271 FITC F7178 Dako
CD28 CD28.2 PE 2071 BC
CD45RA ALB11 PE 1834U BC
TCRa/b IP26 PE 12-9986-73 eBio
CD31 5.6E FITC 1431U BC
CD127 R34.34 PE 1980U BC
CD25 2A3 APC 340907 BD
CXCR5 51505 PE FAB190P R&D

Provider abbreviations: BD: BD-Biosciences; BC: Beckman-Coulter; Dako: DakoCytomation; eBio: eBioscience ; R&D: R&D Systems; * reagents of other manufacturer’s may be used after testing

Required Time

2 hours


Preparation of cells

Whole blood staining does not require special preparation.


Whole blood staining according to the OptiLyse B-method  (Beckman-Coulter).

Step 1: Prepare 15µl of antibody mixture as listed in table below.

Step 2: 50µl whole blood are added, vortexed and incubated for 15 min at room temperature.

Step 3: After thorough mixing, 50µl of Optilyse B are added to each sample, mixed again thoroughly and incubated for 15 min at room temperature.

Step 4: Samples are mixed, 500µl deionised water are added, followed by further incubation for 20 min.

Table Antibody mixtures for the T-cell panel 1)

FITC 200µl PE 200µl PerCP 100µl APC 20µl
T1 CD45RO CXCR5 1:8 CD3 CD4
T2 CD31 CD45RA 1:2 CD3 CD4
T3 CD8 TCRab 1:2 CD3 CD4
T4 CD27 1:5 CD28 1:2 CD3 CD8

1)15µl/50µl whole blood

Stock solution, can be kept at 4°C

Data acquisition

Cells in gate R1 defining the lymphocyte population are displayed in a window FL3 vs. SSC containing a region R2 that includes all CD3+ T cells. Combined regions R1 and R2 define the “T-cell” gate. A third window depicts FL1 versus FL2 and is confined to presentation of T cells.

Suggested Acquisition setting: Events saved: 10000 within the „T-cell“ gate; time limit of 5 min.. It is recommended to record all cells, or at least all lymphocytes, in order to re-examine the specificity of unexpected results with non-T cells.

Total T Cells Total T Cells
CD4+ T Cells CD4+ T Cells
Naive CD4+ T Cells Naive CD4+ T Cells
Memory CD4+ T Cells Memory CD4+ T Cell[...]
CXCR5+CD4 T Cells CXCR5+CD4 T Cells
Recent Thymic Emigrants Recent Thymic Emig[...]
Regulatory CD4 T Cells Regulatory CD4 T C[...]
Naive CD8 T Cells Naive CD8 T Cells
Early Effector CD8 T Cells Early Effector CD8[...]
Late Effector CD8 T Cells Late Effector CD8 [...]
Double Negative T Cells Double Negative T [...]
Click on image to enlarge

Data analysis

First CD4 T cells are gated by setting a region on CD4+ cells within the “T cell gate”. CD4 T cell subpopulations using lymphocyte gate (R1), CD3 (R2) and CD4 (R3) as the line-specific markers are analyzed according to examples displayed in the windows above.

Using the “CD4 T-cell” gate, next T1 and T2 are analysed with quadrant statistics. Panel T1 separates CD45RO+ memory CD4 T cells and the CXCR5+CD45RO+ CD4 T cells. Panel T2 allows the identification of CD45RA+ naive CD4 T cells and of CD31+CD45RA+ recent thymic emigrants.

For the identification of double negative T cells (dnT cells) using the “T cell gate” in panel T3 it is necessary to gate on TCRab+ T cells (R3) in order to exclude g/d T cells. CD3+TCRab+ T cells are then diplayed according to the expression of CD4 and CD8 allowing the identification of CD4 CD8 dn T cells. Panel T4 allows the distinction of different CD8 subpopulations by gating on CD8 (R3) positive cells within the “T cell gate”. The analysis by quadrant statistics displays CD3+CD8+CD27+CD28+ naïve, CD3+CD8+CD27+CD28- early effector and CD3+CD8+CD27-CD28- late effector CD8 T cells.

Data interpretation

The interpretation of the results requires the awareness of the medical history and age of the patient. The reported normal ranges are only reference values for adult patients. In children age related reference values need to be considered. Unexpected results need to be confirmed.


Reported T-cell subpopulations are listed in the table below. Total T-cells as given as percent of lymphocytes, CD4 and CD8 T cells are also given as percentage of lymphocytes, while all CD4 or CD8 T cell subpopulations as recorded percent of the CD4 or CD8 T cells respectively. Absolute counts can be determined immediately when The calcuIation of absolute counts can be performed immediately if absolute lymphocyte count is determined.

Table T-cell subpopulations in peripheral blood

T cell population Reference range
CD3+ of lymphocytes CD3+ T cells 55-83% (1)
CD4+ of lymphocytes CD4+ T cells 28-57% (1)
CD8+ of lymphocytes CD8+ T cells 10-39% (1)
CD4-CD8- of CD3+alpha/beta TCR+ T cells Double negative T cells (DNT) 0.4-2.2%
CD4+CD45RO+ of CD4+ T cells CD4+ memory T cells 29-63%
CD4+CD45RO+ CXCR5+ of CD4+ T cells Follicular like CD4+ T cells 4.6-13%
CD4+CD45RO+ CD127lo CD25+ of CD4+ T cells Treg 1.4-5.1%
CD4+CD45RA+ of CD4+ T cells CD4+ naïve T cells 32-73%
CD4+CD45RA+CD31+ of CD4+CD45RA+ T cells CD4+CD31+ naïve T cells 41-79%
CD8+CD27+CD28+ of CD8+ T cells CD8+ naïve T cells 43-91%
CD8+CD27+CD28- of CD8+ T cells Early CD8+ effector/memory T cells 3-18%
CD8+CD27-CD28- of CD8+ T cells Late CD8+ effector/memory T cells 1-41%

(1) Comans-Bitter et al. (1997) J Pediatr 130, 388-393            (5.-95. Perzentile)

Quality control

Beside standard methods of quality control, a healthy control or commercially available control blood should be run with T-cell stainings of peripheral blood to eliminate staining errors in strongly deviating subpopulations. This is particularly important after production of fresh antibody mixtures.

Trouble shooting

Increased fluorescence of negative subpopulation probably stems from a too high concentration of antibodies. Poor separation of negative and positive populations on the other hand can be caused by overly diluted antibodies. These probable causes should be remembered particularly when using antibodies other than described in this protocol. Monoclonal antibodies or antisera from different manufacturers need to be tested previously.

Need of repetitive evaluation



Michael Schlesier and Klaus Warnatz, CCI University Medical Center Freiburg, Germany
Last update: 2010-04-01 14:48:01

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