1ml EDTA-anti-coagulated or heparinized blood

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

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.

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.

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.

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.

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.

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.

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