Protocol - Characterization of T cell subsets and DC cell subsets for immunophenotyping.
A set of multiplexed fluorescent antibodies was used to characterize the major leukocyte cell populations in peripheral blood, including monocytes, granulocytes, circulating dendritic cells and lymphocytes subdivided into NK, B and T cells and their subsets.
Figure legend:
The Figure 1 illustrates the gating strategy for the T-B-NK, regulatory T cell, maturation stages of T cell and circulating dendritic cell panels on randomly selected blood samples.
For details see supplementary Figure 1 legend of the manuscript by Orrù et al (2013). See citation
Steps
| Description | Temperature | Time | Note |
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Reconstitute Lyophilized antibody cocktail (see table 1 for details) in 100 μl FACS flow buffer (cat # 342003)
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30 minutes
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For Treg, DCs and Maturation of T cells
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Add 100 μl of whole blood by reverse pipetting (to ensure an accurate dispensation)
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Vortex briefly and gently.
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Incubate @ RT protected from light
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15 minutes
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Add 3 ml of lysing solution (BD FACS lysing cat #347691)
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Gently shake by rotation
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30 minutes
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Spin 350g 5 minutes @ RT
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5 minutes
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Pour off supernatant and dry the residual liquid damping the tube edge with a clean tissue
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Add 500 μl of FACS buffer and run the sample onto the cytometer
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This is a lyse-and-wash protocol.
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For DCs actual counts, add 50ul of Liquid Counting Beads (cat #349480) to the samples by reverse pipetting.
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Add 50 μl of whole blood by reverse pipetting
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(to ensure an accurate dispensation)
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Add antibodies (20 ul TBNK cocktail + 5ul CD14 + 5ul HLA-DR + 1ul TCRγδ)
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For TBNK panel
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Vortex briefly
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Incubate @ RT protected from light
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15 minutes
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Add 1 ml of lysing solution (BD FACS lysing cat #347691)
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Vortex briefly
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Incubate @ RT protected from light
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15 minutes
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Run stained sample after 3 hours
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This is a lyse-non-wash protocol to avoid cellular loss
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Other informations
T-B-NK immunophenotype is assessed through the use of standard liquid antibodies while Treg cells, Dendritic Cells and the maturation stages of T cells were assessed by lyotube technology.
i) T-B-NK is a ten parameter and nine antibody panel which was designed to gain a broad reading of leukocytes (CD45+), focusing on lymphocyte subpopulations and dividing them based on CD3 expression. CD3- cells were divided into B cells (CD19+) and natural killer cells (CD16+ or CD56+), while CD3+ cells, i.e. T lymphocytes, were split into 6 subsets based on expression of the CD4 and CD8 markers: CD4- CD8-, CD4- CD8dim (CD8dim), CD4- CD8bright (CD8br), CD4+ CD8br, CD4+ CD8dim, CD4+ CD8- (CD4+). CD8+ corresponds to the summation of CD8bright and CD8dim cells. The extent of T and NK cell activation was detected by the HLA-DR marker which is also constitutively expressed on antigen-presenting cells such as B cells and dendritic cells (DCs). Granulocytes and CD14+ monocytes were also evaluated. In order to get absolute cell counts, Lyse-No-Wash protocol (see below) and BD TruCount™ absolute counting tubes is used for this panel.
ii) Regulatory T cell (Tregs) panel comprises ten parameters and eight antibodies. It identifies CD4+ Tregs according to their high expression of CD25 and low expression of CD127 surface antigens (CD25hi CD127low). Tregs are further subdivided into activated (CD25+++ CD45RA-), resting (CD25++ CD45RA+) and secreting not suppressive (CD25++ CD45RA-). The panel also analyses not regulatory CD4+ CD25hi T cells (CD25hi CD4+ not Tregs), by assessing CD4+ T cells expressing high levels of CD25 and subtracting the Treg cells from them; the resulting population is further divided based on CD45RA positivity. Moreover, the CD8 T cells are divided according to their expression of CD28 and CD45RA antigens. Finally, the panel evaluates the number of Treg subsets, CD4+ T cells and CD8+ T cells expressing the CD39 marker. Staining is performed using a Lyse-Wash (see below) protocol and BD Lyotube™ technology: a cocktail of pre-mixed and pre-titrated fluorescent antibodies directly lyophilized into the acquisition tube. Absolute cell counts are calculated taking into account the absolute number of CD3+ lymphocytes evaluated in the T-B-NK panel.
iii) Maturation stages of T cell panel is characterized by seven parameters and five antibodies. The maturation state of the six T cell subsets, described in the T-B-NK panel (CD4- CD8-, CD4- CD8dim, CD4- CD8bright, CD4+ CD8+, CD4+ CD8dim, CD4+ CD8-), is assessed according to expression of the CD45RA and CCR7 antigens. In each subset naïve (CD45RA+ CCR7+), central memory (CCR7+ CD45RA-), effector memory (CD45RA- CCR7-) and terminally differentiated (CCR7- CD45RA+) maturation stages are analyzed. Staining is done using a Lyse-Wash protocol (see below) and BD Lyotube™ technology. Absolute cell counts are calculated taking into account the absolute number of CD3+ lymphocytes evaluated in the T-B-NK panel.
iv) Circulating Dendritic Cell (cDC) panel includes ten parameters and thirteen antibodies. cDCs are identified based on their brightness for the HLA-DR surface molecule and their negativity for the Lineage cocktail (Lin) targeting CD3, CD14, CD16, CD19, CD20, CD56 markers. The cDCs are classified as myeloid and plasmacytoid based on their positivity for CD11c and CD123 antigens, respectively. Their maturation status is ascertained in relation to the level of the adhesion molecule CD62L, the chemokine receptor CCR2 and of the co-stimulatory molecules CD86 and CD80. Staining is done using a Lyse-Wash protocol (see below) and BD Lyotube™ technology. The absolute number of cells is estimated by adding BD Liquid Counting Beads (cat #349480) to the samples.
Validation info
The protocol has been used to produce data published in peer-reviewed journals.
Funded by: FaReBio2011 “Farmaci e Reti Biotecnologiche di Qualità”
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Table Ab clones.xlsx
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