MBTP 19: Immunophenotyping of regulatory
T cells from mouse spleen using flow cytometry

Miltenyi Biotec-tested panel 19 (MBTP 19)

This application protocol describes the flow cytometric analysis of regulatory T cells (Tregs) after spleen dissociation from healthy C57BL/6 mice. Mouse spleens are easily dissociated using the gentleMACS™ Dissociators to quickly obtain viable single cell suspensions ready for flow cytometric analysis.

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Protocol

Staining protocol

Labeling of cells with Viobility™ Fixable Dye

Viobility Fixable Dye was reconstituted in advance. The vial was warmed up to room temperature to avoid water condensation. One vial of lyophilized Viobility Fixable Dye was reconstituted by addition of 100 µL anhydrous DMSO to the dye vial and mixed until completely dissolved. The solution was aliquoted and stored at –20 °C under anhydrous (desiccant) conditions and protected from light for up to one month.
Volumes given below were for up to 10⁷ nucleated cells. When working with fewer than 10⁷ 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 2×10⁷ nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).

Cell number was determined.
Cells were washed by addition of 1 mL of 1× PBS and centrifuged at 300×g for 10 minutes. Supernatant was aspirated completely.
Cells were resuspended in 100 µL of 1× PBS.
1 µL of Viobility Fixable Dye was added.
Samples were mixed well and incubated for 15 minutes in the dark at room temperature.
▲Note: Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice requires increased incubation times.
Cells were washed by addition 1 mL PEB buffer and centrifuged at 300×g for 10 minutes. Supernatant was aspirated completely.
Step 6 was repeated.
Surface staining was performed.

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

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 FoxP3 Antibody data sheet.

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 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 1× Permeabilization Buffer and the corresponding volume of FoxP3 Antibody 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 1× Permeabilization Buffer and flow cytometric analysis was performed using the MACSQuant® Analyzer.

Flow cytometric analysis

Gating strategy showing the analysis of regulatory T cells (Tregs) from mouse spleen. Splenocytes from C57BL/6 mice were stained using the previously described Treg panel to analyze regulatory T cells. Samples were initially gated on single cells using FSC-H/FSC-A gating and on live cells using Viobility-negative gating (data not shown). Tregs represent a small population of CD3⁺ CD4⁺ T cells (A), that characteristically expresses high levels of CD25 (B, C). CD3⁺ CD4⁺ CD25⁺ T cells express the transcription factor forkhead/winged-helix transcriptional regulator (FoxP3) (G). Moreover, CD3⁺ CD4⁺ T cells can be directly gated on FoxP3 (D) and CD357 (E). A subpopulation of CD4⁺ FoxP3⁺ Tregs also expresses CD134 (F).

Materials

Reagent and instrument requirements

Antibody panel details

Specificity	Clone	Purpose	Fluorochrome	Detection filter (nm) (laser)
CD4	REA604	T helper cells	VioBlue®	450/50 (violet)
Viobility™ 405/520 Fixable Dye	-	Viability	-	525/50 (violet)
FoxP3	REA788	Regulatory T cells	Vio® B515	525/50 (blue)
CD357 (GITR)	REA980	Regulatory T cells	PE	585/40 (blue)
CD3e	REA606	T cells	PE-Vio® 615	655-730 (blue)
CD25	REA568	Regulatory T cells	PE-Vio 770	750LP (blue)
CD134 (OX40)	REA625	Regulatory T cells	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
FoxP3 Staining Buffer Set (# 130-093-142)
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)

Trademarks and disclaimers

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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MBTP 19: Immunophenotyping of regulatory
T cells from mouse spleen using flow cytometry

Miltenyi Biotec-tested panel 19 (MBTP 19)

Protocol

Materials

Protocol

Staining protocol

Labeling of cells with Viobility™ Fixable Dye

Surface staining

Intracellular staining

Flow cytometric analysis

Materials

Reagent and instrument requirements

Antibody panel details

Additional reagents used

Trademarks and disclaimers

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Careers

About Miltenyi Biotec

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MBTP 19: Immunophenotyping of regulatory T cells from mouse spleen using flow cytometry

Miltenyi Biotec-tested panel 19 (MBTP 19)

Protocol

Materials

Protocol

Staining protocol

Labeling of cells with Viobility™ Fixable Dye

Surface staining

Intracellular staining

Flow cytometric analysis

Materials

Reagent and instrument requirements

Antibody panel details

Additional reagents used

Trademarks and disclaimers

Shop

Careers

About Miltenyi Biotec

MBTP 19: Immunophenotyping of regulatory
T cells from mouse spleen using flow cytometry