B cell

IL-4IL-5IL-10IL-13

Antibody production

CD4+ T

Th2

Th17

Th1 Th1

Treg

CD4

TGF-βIL-12

IL-4

IL-17

IL-6+

TGF-β

Lysosome

MHC class II

MHC class II tetramers can be used for direct detection of antigen-speci�c CD4+ T cells.

Biotinylated MHC/peptide complexes are tetramerized with fluorescently-labeled streptavidin, increasing the avidity of MHC/peptide complexes binding to TCR and allowing for direct detection of antigen-specific T cells.

I-Ab MOG35-55 tetramer-PEExperimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis induced by immunization with MOG35-55 peptide, is a widely used experimental model of autoimmune disease. It has been reported that regulatory T (Treg) cells and Th17 cells are involved in the onset of disease, suggesting that the immune balance of CD4+ T cells is important in EAE pathogenesis.

C57BL/6 mice were immunized intraperitoneally twice with 100 nmol of I-Ab-restricted MOG35-55 peptide (MEVGWYRSPFSRVVHLYRNGK, MBL code no. TS-M704-P) and 10 μg cholera toxin (MBL code no. RK-01-511) in complete Freund's adjuvant. Eleven days later, splenocytes were isolated and stained with I-Ab MOG35-55 tetramer-PE (MBL code no. TS-M704-1; Day 0). An aliquot of splenocytes was stimulated with 10μg/mL MOG35-55 peptide for 6 days in vitro and then stained with MHC class II tetramer (Day 6). In vitro stimulation of with I-Ab MOG35-55 resulted in increased tetramer-positive CD4+ T cells from immunized (mouse 1 and 2), but not naïve (mouse 3), mice.

Mouse MHC class II Tetramer

Dendritic cell

MHC Tetramer

mouse 1

mouse 3

mouse 2

CD4 (GK1.5)-FITC CD4 (GK1.5)-FITC

Non-immunized splenocytes

MOG35-55 immunized splenocytes

In vitro MOG35-55

peptide stimulation

MOG35-55 immunized splenocytesDay 0

I-Ab M

OG

35-5

5 Tet

ram

er-P

E

0.17 % 0.22 %

0.05 %

mouse 1

mouse 3

mouse 2

Non-immunized splenocytes

Day 6

I-Ab M

OG

35-5

5 Tet

ram

er-P

E

13.4 % 9.43 %

0.05 %

Code no.

TS-M704-1

TS-M704-P

Product

T-Select I-Ab MOG35-55 Tetramer-PE

I-Ab MOG35-55 Peptide

Size

20 tests

100 μL (10 mg/mL)

Code no.

D341-4

Product

Anti-CD4 (Mouse) mAb-FITC

Size

1 mL (100 tests)

Clone

GK1.5

Isotype

Rat IgG2bκ

MHC

Peptide

Fluorochrome

TCRAntigenspecificT cells

β-chain

α-chain

For Research Use Only

http://ruo.mbl.co.jp/

I-Ab OVA323-339 tetramer-PEOVA is a model antigen commonly used to study immune responses in mice. OT-II transgenic mice express TCR specific for OVA323-339 epitope (ISQAVHAAHAEINEAGR) in the context of I-Ab and serve as an important tool to study differentiation and activation of CD4 T cells. I-Ab OVA323-339 tetramer can be used to monitor antigen-specific CD4 T cell responses in OT-II mice and in various experimental systems using OT-II cells for adoptive transfer.

Reactivity of I-Ab OVA323-339 tetramer reagent was assessed using freshly isolated spleen cells from OT-II mice. The majority of CD8 T cells were tetramer positive in mouse 2, but not in mouse 1 (Figure 1-1). In vitro stimulation with specific peptide significantly increased the number of tetramer positive cells (Figure 1-2).

■ Figure 2: I-Ab OVA323-339 tetramer staining of splenocytes from peptide-immunized mice

OVA-specific CD4 T cells can also be induced in wild-type mice. C57BL/6 mice were immunized twice IP with a mixture of 100 nmol of OVA323-339 peptide and 10 μg of cholera toxin emulsified with adjuvant. After 11 days, spleen cells were harvested and cultured with a final concentration of 1 μg/mL OVA323-339 peptide for 8 days. Cells were tested on days 0 and 8 of culture for OVA tetramer reactivity. In mice immunized with OVA323-339 peptide, induction of specific T cells by in vitro peptide stimulation was confirmed using I-Ab OVA323-339 tetramer. I-Ab MOG35-55 tetramer, used as a negative control, showed no specific staining.

OVA323-339 peptide immunized mouse splenocytes

Day 0 Day 8

I-Ab O

VA32

3-33

9

Tetra

mer

-PE

0.5 % 1.4 %

I-Ab M

OG

35-5

5 Te

tram

er-P

E

0.2 % 0.2 %

CD4 (GK1.5)-FITC

Code no.

TS-M710-1

TS-M703-P

Product

T-Select I-Ab OVA323-339 Tetramer-PE

I-Ad OVA helper peptide

Size

20 tests

100 μL (10 mg/mL)

Tetra

mer

-PE

I-Ab MOG35-55 Tetramer

1.2 %

I-Ab OVA323-339 Tetramer

1.9 %

OVA323-339 peptide

concentration

0 μg /mL

1 μg /mL

1.5 % 79.2 %

CD4 (GK1.5)-FITC

I-Ab O

VA32

3-33

9 Te

tram

er-P

E

mouse 16.4 %

mouse 275.2 %

CD4 (GK1.5)-FITC

I-Ab M

OG

35-5

5 Te

tram

er-P

E

0.5 %

CD4 (GK1.5)-FITC

< mouse 1 >

■ Figure 1-1: I-Ab OVA323-339 tetramer staining of freshly isolated OT-II splenocytes

■ Figure 1-2：I-Ab OVA323-339 tetramer staining of peptide-stimulated OT-II splenocytes (Day 6)

Antibody recognizing Mouse I-Ad / OVA323-339 specific TCR Anti-Mouse TCR DO11.10 (clone KJ1.26, MBL code no. K0221-5) is an antibody that binds to the TCR specific for OVA323-339 presented in the context of I-Ad. This antibody can be used to identify OVA323-339-specific CD4 T cells derived from I-Ad mice. OVA323-339 peptide (ISQAVHAA-HAEINEAGR, MBL code no. TS-M703-P) is often used as a helper peptide for induction of antigen-specific CTL in I-Ad-expressing mice.

■ Flow cytometry with Anti-mouse TCR DO11.10 mAb

Clear Back blocking reagent for tetramer stainingWhen a very rare cell population is detected by a tetramer reagent, it is important to reduce non-specific and Fc-specific staining. Clear Back (MBL code no. MTG-001) is a reagent that blocks non-specific binding to both human and murine cells in flow cytometry and fluorescence microscopy experiments. As shown in Figure 1, Clear Back reduced Fc-specific staining of monocytes by a mouse IgG2a. Clear Back also reduced background staining of peripheral blood mononuclear cells (PBMC) with HLA-A*24:02 EBV BRLF1 tetramer in the tetramer-positive CD8-negative region (Figure 2).

■ Effect of Clear Back on PBMCs

0.3 %0.0 %

FL2

coun

ts

isotypic controlAnti-mouse TCRDO11.10

Anti-

mou

se T

CR

D

O11

.10-

PE

CD4 (GK1.5)-FITCCD4 (GK1.5)-FITC

Isot

ypic

con

trol-P

E

Sample: DO11.10 T cell hybridoma Sample: OVA323-339 peptide immunized BALB/c mouse splenocytes

I-A alleles of mouse strains:I-A allele

Mouse strains

I-Ak

C3H/He

I-Ab

C57BL/-,BXSB/Mp,

129/-

I-Ad

BALB/c,DBA/2

I-As

SJL/J,B10.S

Code no.

K0221-5

Product

Anti-TCR DO11.10 (Mouse) mAb–PE

Isotype

Mouse IgG2aκ

Clone

KJ1.26

Size

1 mL (50 tests)

Code no.

MTG-001

Product

Clear Back (Human Fc receptor blocking reagent)

Size

1 mL (50 tests)x2

SSC

-Hco

unts

coun

ts

FSC-H

FL1-H FITC FL1-H FITC

R1R2

PBMCs were stained with mouse IgG2a-FITC(5 μg/mL)

Red : Clear Back treatmentGreen : No Clear Back treatment

Lymphocytes (R1) Monocytes (R2)

Non-specific binding of antibody was not detected on lymphocyte (R1). Clear Back treatment reduced unwanted Fc-specific staining of monocyte (R2) by a mouse IgG2a.

Tetra

mer

-PE

CD8 (T8)-FITC

Clear Back without Clear Back

Clear Back treatment reduced non-specific binding of a tetramer reagent to tetramer-positive CD8-negative cell population.

Figure 1 Figure 2

Antigen

MOG35-55

FMLV

Eα52-68

ESAT-6

OVA

MHC

I-Ab

I-Ab

I-Ab

I-Ab

I-Ab

Sequence

MEVGWYRSPFSRVVHLYRNGK

EPLTSLTPRCNTAWNRLKL

ASFEAQGALANIAVDKA

MTEQQWNFAGIEAAASAIQG

ISQAVHAAHAEINEAGR

Location(aa)

35-55

123-141

52-68

1-20

323-339

Peptide100 μL (10 mg/mL)

TS-M704-P

—

—

TS-M707-P

TS-M703-P

PE-labeled20 tests

TS-M704-1

TS-M705-1

TS-M706-1

TS-M707-1

TS-M710-1

APC-labeled20 tests

■ Mouse MHC class II Tetramer

■ Mouse class II epitope peptide

T-Select Mouse MHC class II Tetramers & related products

Contact us

Antigen

OVA

OVA E1

OVA G4

OVA Q4H7

MHC

H-2Kb

H-2Kb

H-2Kb

H-2Kb

Sequence

SIINFEKL

EIINFEKL

SIIGFEKL

SIIQFEHL

Location(aa)

257-264

257-264

257-264

257-264

Peptide100 μL (10 mg/mL)

TS-5001-P

—

—

—

PE-labeled50 tests

TS-5001-1C

TS-M541-1

TS-M542-1

TS-M543-1

APC-labeled50 tests

TS-5001-2C

TS-M541-2

TS-M542-2

TS-M543-2

■ Mouse MHC class I OVA Tetramer

Product

T-Select Mouse CD1d Tetramer

PE-labeled50 tests

TS-MCD-1

PE-labeled10 tests

TS-MCD-1S

APC-labeled50 tests

TS-MCD-2

■ Mouse CD1d Tetramer

Origin

HBc

TT p30

OVAMOG

ESAT-6

HEL

MHC

I-Ab

I-Ad

I-Ab, I-Ad

I-Ab

I-Ab

I-Ak

Size

1 mg

1 mg

1 mg

1 mg

1 mg

1 mg

Sequence

TPPAYRPPNAPIL

FNNFTVSFWLRVPKVSASHLE

ISQAVHAAHAEINEAGR

MEVGWYRSPFSRVVHLYRNGK

MTEQQWNFAGIEAAASAIQG

DGSTDYGILQINSRW

Location（aa）

128-140

947-967

323-339

35-55

1-20

48-62

Code no.

TS-M701-P

TS-M702-P

TS-M703-P

TS-M704-P

TS-M707-P

TS-M708-P

■ AntibodyClone

GK1.5

KJ1.26

KJ1.26

KJ12.98

3G8

6H3

6H3

Size

1 mL (100 tests)

100 μg

1 mL (50 tests)

100 μg

50 μg/1 mL

100 μg/100 μL

10 μg/1 mL

Product

Anti-CD4 (Mouse) mAb-FITC

Anti-TCR DO11.10 (Mouse) mAb

Anti-TCR DO11.10 (Mouse) mAb–PE

Anti-TCR 3DT-52.5 (Mouse) mAb

Rat IgG2b (isotype control)-FITC

Mouse IgG2a (isotype control)

Mouse IgG2a (isotype control)-PE

Isotype

Rat IgG2bκ

Mouse IgG2aκ

Mouse IgG2aκ

Mouse IgG2aκ

Rat IgG2bκ

Mouse IgG2aκ

Mouse IgG2aκ

Code no.

D341-4

K0221-3

K0221-5

K0222-3

M090-4

M076-3

M076-5

■ ReagentSize

150 tests

1 mL (50 tests)x2

250 tests

200 tests

Product

7-AAD Viability Dye

Clear Back (Human Fc receptor blocking reagent)

OptiLyse B

OptiLyse C

Code no.

A07704

MTG-001

IM-1400

A11895

■ KitSize

50 tests

Product

IMMUNOCYTO Cytotoxity Detection Kit

Code no.

AM-1005

146064-140512014.05

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