Immunophenotyping of T cells from mouse spleen using flow cytometry

Miltenyi Biotec-tested panel 11 (MBTP 11)

This application protocol describes the flow cytometric analysis of major T cell subsets after spleen dissociation from healthy C57BL/6 mice. Mouse spleens can easily be dissociated using the gentleMACS™ Dissociators to quickly obtain viable single cell suspensions ready for downstream applications, such as flow cytometric analysis.

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Protocol

Flow cytometry 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.
Cells were resuspended in 100 µL of PEB buffer and flow cytometric analysis was performed using the MACSQuant® Analyzer.

Flow cytometric analysis

Gating strategy showing the analysis of T cell subsets from mouse spleen. Splenocytes from C57BL/6 mice were stained using the previously described T cell panel to identify major T cell subsets. CD4⁺ and CD8⁺ T cells were identified from CD3-expressing cell subsets (A, B). Different subsets of naive, central memory (CM), effector memory (EM), CD127-, and KLRG1-expressing T cells were analyzed within CD4⁺ (C, D, E) and CD8⁺ T cells (F, G, H) respectively. The differential expression of the adhesion molecules CD62L and CD44 was used to identify naive T cells from CM and EM T cells. Naive T cells expressed high levels of CD62L and showed absent or low expression of CD44 (C). Both CM and EM populations expressed CD44. CM T cells also expressed CD62L, whereas expression of CD62L was absent in EM T cells. Analysis of CD127 and KLRG1 was included in this panel to allow the study of T cell responses during infection. During the course of an infection the levels of KLRG1 increase, especially on CD8⁺ T cells.

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)
CD62L	REA828	Memory T cells	FITC	525/50 (blue)
CD127	REA680	Infection response	PE	585/40 (blue)
CD3e	REA606	T cells	PE-Vio® 615	655-730 (blue)
CD44	REA644	Memory T cells	PE-Vio 770	750LP (blue)
KLRG1	REA1016	Infection response	APC	655-730 (red)
CD8a	REA601	Cytotoxic T cells	APC-Vio 770	750LP (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
Flow cytometer, for example, MACSQuant® Analyzer 10 (# 130-096-343) or MACSQuant X (# 130-105-100)
Viobility™ 405/520 Fixable Dye (130-109-814)
(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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Immunophenotyping of T cells from mouse spleen using flow cytometry

Miltenyi Biotec-tested panel 11 (MBTP 11)

Protocol

Materials

Protocol

Flow cytometry staining protocol

Labeling of cells with Viobility™ Fixable Dye

Surface staining

Flow cytometric analysis

Materials

Reagent and instrument requirements

Antibody panel details

Additional reagents used

Trademarks and disclaimers

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Immunophenotyping of T cells from mouse spleen using flow cytometry

Miltenyi Biotec-tested panel 11 (MBTP 11)

Protocol

Materials

Protocol

Flow cytometry staining protocol

Labeling of cells with Viobility™ Fixable Dye

Surface staining

Flow cytometric analysis

Materials

Reagent and instrument requirements

Antibody panel details

Additional reagents used

Trademarks and disclaimers

Shop

Careers

About Miltenyi Biotec