MBTP 26: Immunophenotyping of regulatory T cells from human PBMCs using flow cytometry

Miltenyi Biotec-tested panel 26 (MBTP 26)

This application protocol describes the simultaneous quantification of intracellular and surface markers for easy enumeration of regulatory T cells from human PBMCs using flow cytometry.

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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.
A master mix with all antibodies was prepared according to the dilutions mentioned in the respective antibody data sheets.
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.

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 per 10⁶ cells 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 10 µL of Tandem Signal Enhancer and 88 µL of cold 1× Permeabilization Buffer.
2 µL of FoxP3 Antibody, anti-human, Vio® 667, REAfinity™ was added to the cells.
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 PEB buffer and flow cytometric analysis was performed using the MACSQuant® Analyzer.

Gating strategy showing the analysis of human regulatory T cells. Human PBMCs were stained for regulatory T cell markers using the previously described panel. CD45⁺ cells were gated as P1 (A). From P1, autofluorescent cell debris was excluded as P2 (B). From P2, CD4⁺ cells were gated as P3 (C). From P3, regulatory T cells were identified by analysis of CD25, CD127, and FoxP3 expression (D–F).

Materials

Reagent and instrument requirements

Antibody panel details

Specificity	Clone	Purpose	Fluorochrome	Detection filter (nm) (laser)
CD45	REA747	Lymphocytes	VioBlue®	450/50 (violet)
CD4	REA623	T helper cells	VioGreen™	525/50 (violet)
CD25	REA570	Regulatory T cells	Vio® Bright B515	525/50 (blue)
CD127	REA614	Regulatory T cells	PE	585/40 (blue)
FoxP3	REA944	Transcription factor of regulatory T cells	Vio 667	655-730 (blue)

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
Treg Detection Kit, human (# 130-122-994)
(Optional) Transcription Factor Staining Buffer Set (# 130-122-981)
(Optional) Tandem Signal Enhancer, human (# 130-099-887)
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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Matching products:

130-093-142

FoxP3 Staining Buffer Set

for 100

tests

(1)

EUR 221,00

130-093-607

MACSQuant
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Calibration Beads

3.5 mL

EUR 239,00

130-096-343

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130-099-887

Tandem Signal Enhancer, human

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X

unit

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130-104-693

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Comp Bead Kit, anti-REA

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130-110-637

CD45 Antibody, anti-human, VioBlue
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in 200 µL (1)

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130-110-775

CD45 Antibody, anti-human, VioBlue
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REA747

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130-113-976

CD127 Antibody, anti-human, PE, REAfinity™

REA614

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in 60 µL (1)

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CD4 Antibody, anti-human, VioGreen™, REAfinity™

REA623

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CD25 Antibody, anti-human, Vio
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REA570

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Transcription Factor Staining Buffer Set

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EUR 177,00

MBTP 26: Immunophenotyping of regulatory T cells from human PBMCs using flow cytometry

Miltenyi Biotec-tested panel 26 (MBTP 26)

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

FoxP3 Staining Buffer Set

MACSQuant
^®
Calibration Beads

MACSQuant
^®
Analyzer 10

Tandem Signal Enhancer, human

MACSQuant
^®
X

MACS
^®
Comp Bead Kit, anti-REA

CD45 Antibody, anti-human, VioBlue
^®
, REAfinity™

CD45 Antibody, anti-human, VioBlue
^®
, REAfinity™

CD127 Antibody, anti-human, PE, REAfinity™

CD4 Antibody, anti-human, VioGreen™, REAfinity™

CD25 Antibody, anti-human, Vio
^®
Bright B515
, REAfinity™

Transcription Factor Staining Buffer Set

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