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Mouse t activator anti cd3 cd28 dynabeads

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Mouse T-Activator anti-CD3/CD28 Dynabeads are uniform, superparamagnetic beads coated with monoclonal antibodies against mouse CD3 and CD28 molecules. These beads can be used to activate and expand mouse T cells in vitro.

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Lab products found in correlation

Easysep mouse cd4 t cell isolation kit, by STEMCELL (4 mentions) Mouse erythrocyte lysis kit, by R&D Systems (2 mentions) Celltrace violet ctv, by Thermo Fisher Scientific (2 mentions) Easysep mouse cd8 t cell isolation kit, by STEMCELL (2 mentions) Anti il 4 11b11, by BioLegend (2 mentions) Rmil 12 p70, by R&D Systems (2 mentions) Rmtgf β1, by R&D Systems (2 mentions) Robosep buffer, by STEMCELL (2 mentions) Magcellect mouse cd8 t cell isolation kit, by R&D Systems (1 mentions) Interleukin 2 (il 2), by R&D Systems (1 mentions) Mouse cd4 t cell isolation kit, by Thermo Fisher Scientific (1 mentions)

8 protocols using mouse t activator anti cd3 cd28 dynabeads

1

Mouse CD8+ T Cell Isolation and Activation

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Spleens were harvested from age- and strain-matched donor mice, minced, and dispersed into single cells in RPMI-10 (RPMI-1640 with 10% fetal bovine serum) by pressing between sterile frosted glass slides. Cells were filtered through a 40-μm pore-size cell strainer and washed in RPMI-10, and red blood cells (RBCs) were lysed using the mouse erythrocyte lysis kit (catalog no. WL2000, R&D Systems). CD8+ T cells were enriched by negative selection on a MagCellect mouse CD8+ T cell isolation kit (catalog no. MAGM203, R&D Systems) as instructed by the manufacturer. The yield of CD8+ T cells was about 107 per spleen, and 95% of the isolated cells expressed CD8a. The cells were cultured at 1 million/ml of RPMI-10 medium with recombinant mouse interleukin-2 (IL-2) (no. 402-ML, R&D Systems) at 30 IU/ml and CD28/CD3 beads (Mouse T-activator anti-CD3/CD28 Dynabeads, Gibco) that were added at a 1:1 ratio with the CD8+ T cells.
Kansal R., Richardson N., Neeli I., Khawaja S., Chamberlain D., Ghani M., Ghani Q.U., Balazs L., Beranova-Giorgianni S., Giorgianni F., Kochenderfer J.N., Marion T., Albritton L.M, & Radic M. (2019). Sustained B cell depletion by CD19-targeted CAR T cells is a highly effective treatment for murine lupus. Science translational medicine, 11(482), eaav1648.
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2

Evaluating Treg-mediated Suppression of CD8+ T Cells

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Naive CD45.2+CD8+ T responder cells (Tresp) were negatively sorted using the EasySep Mouse CD8+ T Cell Isolation kit (STEMCELL Technologies Inc.) and stained with CellTrace Violet (CTV; Life Technologies, Carlsbad, CA). CD45.1+CD4+FoxP3-EGFP− T cells that had experienced HD Lm ΔactA-Ova infection for 24 h in C57BL/6 mice were re-isolated using the EasySep Mouse CD4+ T Cell Isolation kit (STEMCELL Technologies Inc.) followed by fluorescent cell sorting CD45.1+CD45.2−FoxP3-EGFP+ and CD45.1+CD45.2−FoxP3-EGFP− cells. 2.5 × 104 CD45.2+CD8+ Tresp were placed in co-culture with graded numbers of sex-matched, magnetic/fluorescent cell sorted CD45.1+FoxP3-EGFP+ (Treg) or EGFP− (conventional) CD4+ T cells and 1.25 mL washed Mouse T-Activator anti-CD3/CD28 Dynabeads (GIBCO) in a 96-well round-bottom plate for 72 h. Cell proliferation of CD45.2+CD8+ Tresp was determined by flow cytometry based on dilution of CTV. Percent suppression was calculated as the percent difference in absolute number of dividing Tresp in wells containing EGFP+ or EGFP– CD4+ T cells to the average absolute number of dividing Tresp in wells without addition of EGFP+ or EGFP– CD4+ T cells. Replication index, representative of only Tresp that had responded to anti-CD3/CD28 stimulation, was also determined for the cultures.
Dolina J.S., Lee J., Griswold R.Q., Labarta-Bajo L., Kannan S., Greenbaum J.A., El Idrissi N.B., Pont M.J., Croft M, & Schoenberger S.P. (2020). TLR9 Sensing of Self-DNA Controls Cell-Mediated Immunity to Listeria Infection via Rapid Conversion of Conventional CD4+ T Cells to Treg. Cell reports, 31(1), 107249.
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3

Fli-1 Deletion in T Cells

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Mice with a Fli-1 conditional deletion (Fli-1flox/flox) were described previously (22 (link), 40 (link)). A T-cell conditional deletion of Fli-1 exons 3 and 4 was mediated by a Cre/Lox system utilizing the CD4 promoter (41 (link)). Homozygous Fli-1 deletion in T cells was mediated via Fli-1flox/floxCD4Cre. Wild-type Fli-1 with CD4cre+ were used as controls (referred to as Fli-1+/+CD4cre). Spleens were harvested from Fli-1flox/floxCD4Cre and Fli-1+/+CD4Cre mice at the age of 8-10 weeks, minced, and dispersed into single cells in RPMI 1640 medium with 10% fetal bovine serum. Cells were filtered through a 40-μm pore-size cell strainer and washed with RPMI 1640 medium, and red blood cells (RBCs) were lysed using the mouse erythrocyte lysis kit (R&D Systems, Minneapolis, MN). CD4+ T cells were enriched by negative selection using a mouse CD4+ T cell isolation kit (Invitrogen, Waltham, MA) as instructed by the manufacturer. The cells were cultured at 1 million/mL of RPMI 1640 medium with recombinant mouse interleukin-2 (IL-2) (R&D Systems, Minneapolis, MN) at 20 IU/mL and CD28/CD3 beads (Mouse T-activator anti-CD3/CD28 Dynabeads, Gibco) that were added in a 1:1 ratio with the CD4+ T cells. The supernatants of T cells were collected after three days of stimulation, and GM-CSF cytokine production was measured by ELISA.
Wang X., Richard M.L., Li P., Henry B., Schutt S., Yu X.Z., Fan H., Zhang W., Gilkeson G, & Zhang X.K. (2020). Expression of granulocyte-macrophage colony-stimulating factor is regulated by Fli-1 transcription factor, a potential drug target. Journal of immunology (Baltimore, Md. : 1950), 206(1), 59-66.
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4

Induction of Regulatory T Cells

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Conventional CD4+ T cells were isolated from CD45.1+Foxp3EGFP or CD45.1+Il12rb2−/−Foxp3EGFP mice using the EasySep Mouse CD4+ T Cell Isolation kit (STEMCELL Technologies Inc.) followed by fluorescent cell sorting of EGFP− T cells. 2 3 105 CD4+FoxP3-EGFP− T cells were placed in culture with 5 μL washed Mouse T-Activator anti-CD3/CD28 Dynabeads (GIBCO, Carlsbad, CA) and 1 μg/mL anti-IL-4 (11B11) (BioLegend, San Diego, CA) in a 96-well round-bottom plate for 24 h. Select wells contained 5 ng/mL rmTGF-β1 and 10 ng/mL rmIL-12 (p70) (R&D Systems, Minneapolis, MN). Nuclear FACS staining of EGFP, FoxP3, and T-bet were used to measure conventional CD4+ T cell conversion to phenotypic Treg.
Dolina J.S., Lee J., Griswold R.Q., Labarta-Bajo L., Kannan S., Greenbaum J.A., El Idrissi N.B., Pont M.J., Croft M, & Schoenberger S.P. (2020). TLR9 Sensing of Self-DNA Controls Cell-Mediated Immunity to Listeria Infection via Rapid Conversion of Conventional CD4+ T Cells to Treg. Cell reports, 31(1), 107249.
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5

Evaluating Treg-mediated Suppression of CD8+ T Cells

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Naive CD45.2+CD8+ T responder cells (Tresp) were negatively sorted using the EasySep Mouse CD8+ T Cell Isolation kit (STEMCELL Technologies Inc.) and stained with CellTrace Violet (CTV; Life Technologies, Carlsbad, CA). CD45.1+CD4+FoxP3-EGFP− T cells that had experienced HD Lm ΔactA-Ova infection for 24 h in C57BL/6 mice were re-isolated using the EasySep Mouse CD4+ T Cell Isolation kit (STEMCELL Technologies Inc.) followed by fluorescent cell sorting CD45.1+CD45.2−FoxP3-EGFP+ and CD45.1+CD45.2−FoxP3-EGFP− cells. 2.5 × 104 CD45.2+CD8+ Tresp were placed in co-culture with graded numbers of sex-matched, magnetic/fluorescent cell sorted CD45.1+FoxP3-EGFP+ (Treg) or EGFP− (conventional) CD4+ T cells and 1.25 mL washed Mouse T-Activator anti-CD3/CD28 Dynabeads (GIBCO) in a 96-well round-bottom plate for 72 h. Cell proliferation of CD45.2+CD8+ Tresp was determined by flow cytometry based on dilution of CTV. Percent suppression was calculated as the percent difference in absolute number of dividing Tresp in wells containing EGFP+ or EGFP– CD4+ T cells to the average absolute number of dividing Tresp in wells without addition of EGFP+ or EGFP– CD4+ T cells. Replication index, representative of only Tresp that had responded to anti-CD3/CD28 stimulation, was also determined for the cultures.
Dolina J.S., Lee J., Griswold R.Q., Labarta-Bajo L., Kannan S., Greenbaum J.A., El Idrissi N.B., Pont M.J., Croft M, & Schoenberger S.P. (2020). TLR9 Sensing of Self-DNA Controls Cell-Mediated Immunity to Listeria Infection via Rapid Conversion of Conventional CD4+ T Cells to Treg. Cell reports, 31(1), 107249.
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6

Induction of Regulatory T Cells

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Conventional CD4+ T cells were isolated from CD45.1+Foxp3EGFP or CD45.1+Il12rb2−/−Foxp3EGFP mice using the EasySep Mouse CD4+ T Cell Isolation kit (STEMCELL Technologies Inc.) followed by fluorescent cell sorting of EGFP− T cells. 2 3 105 CD4+FoxP3-EGFP− T cells were placed in culture with 5 μL washed Mouse T-Activator anti-CD3/CD28 Dynabeads (GIBCO, Carlsbad, CA) and 1 μg/mL anti-IL-4 (11B11) (BioLegend, San Diego, CA) in a 96-well round-bottom plate for 24 h. Select wells contained 5 ng/mL rmTGF-β1 and 10 ng/mL rmIL-12 (p70) (R&D Systems, Minneapolis, MN). Nuclear FACS staining of EGFP, FoxP3, and T-bet were used to measure conventional CD4+ T cell conversion to phenotypic Treg.
Dolina J.S., Lee J., Griswold R.Q., Labarta-Bajo L., Kannan S., Greenbaum J.A., El Idrissi N.B., Pont M.J., Croft M, & Schoenberger S.P. (2020). TLR9 Sensing of Self-DNA Controls Cell-Mediated Immunity to Listeria Infection via Rapid Conversion of Conventional CD4+ T Cells to Treg. Cell reports, 31(1), 107249.
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7

CD4+ T Cell Isolation and Culture

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About 6 to 8-week-old MCUfl/fl CD4Cre+ or MCUfl/fl MB1Cre+ mice and control counterparts were humanely sacrificed immediately prior to immune cell isolation. Superficial cervical and inguinal lymph nodes were first collected followed by the spleen. All lymphoid tissues were then disrupted by passing the tissue through a 70-μm mesh screen. The resulting single-cell suspension was than washed with 10 ml of cold RPMI and resuspended in 1 ml of Robosep buffer (STEMCELL technologies) prior to the negative selection of B or T cells. Using the corresponding negative selection kit primary mouse CD4+ T cells and B cells were isolated using the magnetic beads and antibody cocktail as recommended by the manufacturer.
Isolated CD4+ T cells were then cultured ex vivo to increase the efficiency of CREERT2 activity. Naïve primary CD4+ T cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 1× Antibiotic-Antimycotic, 1× Glutamax, mIL2 (30 U/ml), and Mouse T-Activator anti-CD3/CD28 Dynabeads (Thermo Fisher) at a concentration of one bead per cell. Experimental mice (MCUfl/fl CD4Cre+) and control (MCUfl/fl CD4Cre-) mice were both treated with 2 μM 4-OHT for 4 days. As a control, the second group of (MCUfl/fl CD4Cre-) mice was only administered vehicle (dimethyl sulfoxide). Cells were subsequently washed and beads removed.
Yoast R.E., Emrich S.M., Zhang X., Xin P., Arige V., Pathak T., Benson J.C., Johnson M.T., Abdelnaby A.E., Lakomski N., Hempel N., Han J.M., Dupont G., Yule D.I., Sneyd J, & Trebak M. (2021). The Mitochondrial Ca2+ uniporter is a central regulator of interorganellar Ca2+ transfer and NFAT activation. The Journal of Biological Chemistry, 297(4), 101174.
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8

Isolation and Culture of Primary CD4+ T Cells

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Six to eight-week-old MCU fl/fl CD4 Cre+ or MCU fl/fl MB1 Cre+ mice and control counterparts were humanly sacrificed immediately prior to immune cell isolation. Superficial cervical and Inguinal lymph nodes were first collected followed by the spleen. All lymphoid tissues were then disrupted by passing the tissue through a 70 µm mesh screen. The resulting single cell suspension was than washed with 10 mL of cold RPMI and resuspended in 1 mL of Robosep buffer (STEMCELL technologies) prior to the negative selection of B-or T-cells. Using the corresponding negative selection kit primary mouse CD4 + T-cells and B-cells were isolated using the magnetic beads and antibody cocktail as recommended by the manufacturer.
Isolated CD4 + T-cells were then cultured ex vivo to increase the efficiency of CRE ERT2 activity. Naïve Primary CD4 + T were maintained in RPMI 1640 supplemented with 10% FBS, 1X Antibiotic-Antimycotic, 1X Glutamax, mIL2 (30 U/mL), and Mouse T-Activator anti-CD3/CD28 Dynabeads (Thermo Fisher) at a concentration of one bead per cell. Experimental mice (MCU fl/fl CD4 Cre+ ) and control (MCU fl/fl CD4 Cre-) mice were both treated with 2 µM 4-OHT for four days. As a control, the second group of (MCU fl/fl CD4 Cre-) mice was only administered vehicle (DMSO). Cells were subsequently washed and beads removed.
Yoast R.E., Emrich S.M., Zhang X., Xin P., Arige V., Pathak T., Benson J.C., Johnson M., Lakomski N., Hempel N., Han J.M., Dupont G., Yule D.I., Sneyd J., & Trebak M. (2021). Regulation of Interorganellar Ca2+ Transfer and NFAT Activation by the Mitochondrial Ca2+ Uniporter.
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