MBTP 31: Immunophenotyping of human T cells via transcription factor profiling using flow cytometry

Miltenyi Biotec-tested panel 31 (MBTP 31)

This application protocol describes the flow cytometric analysis of the expression of transcription factors by T cell subsets from human PBMCs.

Print protocol

Protocol

Surface staining

Volumes given below were for up to 10⁶ nucleated cells. When working with fewer than 10⁶ cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g. for 2×10⁶ nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).

Cell number was determined.
Cells were centrifuged at 300×g for 10 minutes. Supernatant was aspirated completely.
An antibody master mix was prepared according to the dilutions mentioned in the respective antibody data sheets.
▲Note: Helios, Ki-67 and T-bet are intranuclear markers and will be detected after fixation and permeabilization of cells.
100 µl antibody master mix was added to the cells.
Cells were resuspended and incubated for 10 minutes in the dark at 4 °C.
Cells were washed by addition of 1 mL of PEB buffer and centrifuged at 300×g for 5 minutes at 4 °C. Supernatant was aspirated completely.
Intracellular staining was performed.

Intracellular staining

Volumes given below were for up to 10⁶ nucleated cells. When working with fewer than 10⁶ cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g. for 2×10⁶ nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).
Reagents were prepared freshly. Failure to do so may lead to sub-optimal results.
The required total buffer volumes were calculated beforehand. Volumes depend on the number of cells to be analyzed as well as the number of tests to be performed. For more information, please refer to the data sheet of the Transcription Factor Staining Buffer Set.

Fixation and Permeabilization solution was prepared by diluting 1:3 the Fixation/Permeabilization Solution 1 with the Fixation/Permeabilization Solution 2.
1× solution of Permeabilization Buffer was prepared by diluting the 10× Permeabilization buffer with deionized or distilled water.
▲Note: Before preparing the dilution make sure the buffer does not contain any precipitates.
Cells previously stained for surface markers were resuspended in 1 mL of cold, freshly prepared Fixation and Permeabilization Solution.
Cells were mixed well and incubated for 30 minutes in the dark at 4 °C.
Cells were washed by adding 1 mL of cold PEB buffer and centrifuged at 300×g for 5 minutes at 4 °C. Supernatant was aspirated completely.
Cells were washed by adding 1 mL of cold 1× Permeabilization Buffer and centrifuged at 300×g for 5 minutes at 4 °C. Supernatant was aspirated completely.
Cells were resuspended in 1× Permeabilization Buffer and the corresponding antibody volume according to the dilution mentioned in the respective data sheet was added.
Cells were mixed well and incubated for 30 minutes in the dark at 4 °C.
Cells were washed by adding 1 mL of cold 1× Permeabilization Buffer and centrifuged at 300×g for 5 minutes at 4 °C. Supernatant was aspirated completely.
Cells were resuspended in 100 µL of PEB buffer and flow cytometric analysis was performed using the MACSQuant® Analyzer.

Gating strategy showing the analysis of the expression of transcription factors of T cell subsets from human PBMCs. T cells from human PBMCs were stained using the previously described panel to analyze the expression of transcription factors. Samples were initially gated on single CD45RA⁺ cells (data not shown). T cell subsets were identified by CD4 and CD8 expression (A). Regulatory T cells were discriminated based on CD25 and CD127 expression (B). Expression of the transcription factors Helios, Ki-67, and T-bet was monitored in CD8⁺ T cells (C–E) and in regulatory T cells (F–H).

Materials

Reagent and instrument requirements

Antibody panel details

Specificity	Clone	Purpose	Fluorochrome	Detection filter (nm) (laser)
CD45RA	REA562	Leukocytes	PE-Vio® 615	655-730 (blue)
CD4	REA623	T helper cells	APC-Vio 770	750 LP (red)
CD8	REA734	Cytotoxic T cells	VioGreen™	525/50 (violet)
CD25	REA570	Regulatory T cells	PE	585/40 (blue)
CD127	REA614	Cell type exclusion	PE-Vio 770	750 LP (blue)
Helios	REA829	Transcription factor	VioBlue®	450/50 (violet)
Ki-67	REA183	Transcription factor	Vio B515	525/50 (blue)
T-bet	REA102	Transcription factor	APC	655-730 (red)

Additional reagents used

Phosphate-buffered saline (PBS), pH 7.2
PEB buffer: PBS, pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA.
Deionized or distilled water
Transcription Factor Staining Buffer Set (# 130-122-981)
Flow cytometer, for example, MACSQuant® Analyzer 10 (# 130-096-343) or MACSQuant X (# 130-105-100)
(Optional) MACSQuant Calibration Beads (# 130-093-607)
(Optional) MACS® Comp Bead Kit, anti-REA (# 130-104-693)

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Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use.

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For copyright note and a complete list of trademarks visit www.miltenyibiotec.com/legalnotes.

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MBTP 31: Immunophenotyping of human T cells via transcription factor profiling using flow cytometry

Miltenyi Biotec-tested panel 31 (MBTP 31)

Protocol

Materials

Protocol

Staining protocol

Surface staining

Intracellular staining

Flow cytometric analysis

Materials

Reagent and instrument requirements

Antibody panel details

Additional reagents used

Trademarks and disclaimers

MACSQuant
^®
Calibration Beads

MACSQuant
^®
Analyzer 10

MACSQuant
^®
X

MACS
^®
Comp Bead Kit, anti-REA

CD8 Antibody, anti-human, VioGreen™, REAfinity™

CD8 Antibody, anti-human, VioGreen™, REAfinity™

CD127 Antibody, anti-human, PE-Vio
^®
770
, REAfinity™

CD4 Antibody, anti-human, APC-Vio
^®
770
, REAfinity™

CD25 Antibody, anti-human, PE, REAfinity™

7-AAD Staining Solution

CD45RA Antibody, anti-human, PE-Vio
^®
615
, REAfinity™

Ki-67 Antibody, anti-human/mouse, Vio
^®
B515
, REAfinity™

T-bet Antibody, anti-human/mouse, APC, REAfinity™

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