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T cell activation expansion kit

Manufactured by Miltenyi Biotec
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Sourced in Germany, United States, United Kingdom, France

The T cell activation/expansion kit is a laboratory product designed to activate and expand T cells. The kit provides the necessary components to stimulate T cell proliferation and growth in an in vitro setting.

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Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (6 mentions) Pan t cell isolation kit, by Miltenyi Biotec (5 mentions) Penicillin streptomycin, by Merck Group (3 mentions) Recombinant human tgf β, by PAN Biotech (3 mentions) Automacs, by Miltenyi Biotec (3 mentions) Cd4 t cell isolation kit, by Miltenyi Biotec (3 mentions) Ionomycin, by Merck Group (3 mentions) Countess 2, by Thermo Fisher Scientific (3 mentions) Hiseq 2000, by Illumina (2 mentions) Ifnγ secretion assay cell enrichment and detection kit, by Miltenyi Biotec (2 mentions) Cd8 microbead, by Miltenyi Biotec (2 mentions) Interleukin 6 (il 6), by Miltenyi Biotec (2 mentions) Rpmi 1640 medium, by Merck Group (2 mentions) Recombinant human il 2, by Miltenyi Biotec (2 mentions) X vivo 15, by Lonza (2 mentions) Polycarbonate membrane insert, by Corning (2 mentions) Naive t cell isolation beads, by Miltenyi Biotec (2 mentions) Recombinant il 10, by Thermo Fisher Scientific (2 mentions) Anti il10 clone jes3 19f1, by BD (2 mentions)

Close spelling variants of this product

Mouse T cell Activation and Expansion Kit (one bead/cell; T cell activation/expansion kit Mouse T cell Activation/Expansion Kit MACSiBeads from the T Cell Activation/Expansion Kit Human T Cell Activation/Expansion Kit T cell activation and expansion kit T cell expansion kit T cell activation kit

88 protocols using t cell activation expansion kit

1

Evaluating T Cell Proliferation with CFSE

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Cell proliferation was assessed using the carboxyfluoresceindiacetatesuccinimidyl ester (CFSE) dilution assay. Briefly, CD4+ T cells were stained with CFSE (Invitrogen, 1 µg/mL, 15 min at 37 °C), washed and cultured in RPMI medium supplemented with fetal bovine serum at 37 °C and 5% CO2. Subsequently, T cells were seeded in 96 flat-bottom well plates (Costar Corning, 100,000/well) and stimulated with anti-CD3/anti-CD28 mAb coated beads (T cell activation/expansion kit, Miltenyi Biotec), in the presence or absence of hAEC (at hAEC:effector cell ratios ranging from 1:1 to 1:8). In the control group, hAEC were treated for 30 min with anti-HLA-G (#87G, Exbio, 10 µg/mL), anti-HLA-E (#3D12, Biolegend, 20 µg/mL) or anti-β2 microglobulin (kindly supplied by Dr. Fabio Malavasi, University of Turin, Italy).
In another set of experiments, 100,000 stimulated T cells were cultured in the presence of ssEV or lsEV derived from 40 mL of hAEC supernatant derived from 75 cm2 at confluence (5 millions of cells) and resuspended in 100 µL of culture medium, or with hAEC (at hAEC:effector cell ratio 1:1) previously cultured in the presence of inhibitors of EV release, as described above.
Effector cells were harvested after 6 days, and stained with PE-conjugated anti-CD4 mAb (Beckman Coulter). CFSE/CD4 positive cells were analyzed using the Gallios cytometer and Kaluza software.
Morandi F., Marimpietri D., Görgens A., Gallo A., Srinivasan R.C., El-Andaloussi S, & Gramignoli R. (2020). Human Amnion Epithelial Cells Impair T Cell Proliferation: The Role of HLA-G and HLA-E Molecules. Cells, 9(9), 2123.
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2

Exome Sequencing of Tumor and Constitutional DNA

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For cases E4051 and E6526 we sequenced both tumor (peripheral blood leucocyte) and constitutional (cultured T-cell) DNA. T-cells were isolated from anticoagulated peripheral blood using CD3 MicroBeads and cultured using a T-cell Activation/Expansion kit (Miltenyi Biotec, Bisley, UK). Samples were prepared for exome sequencing using the Agilent SureSelect kit (Agilent Technologies, Palo Alto, CA, USA) (Human All Exon 50 Mb) and then sequenced on an Illumina HiSeq 2000 (Illumina, Great Abington, UK) at the Wellcome Trust Centre for Human Genetics, Oxford, UK. Exome sequencing of ULSAM1182, ULSAM1242 and ULSAM1356 (peripheral blood leukocyte DNA only) was performed by SciLifeLab (Stockholm, Sweden). Sequence data from all five samples were analyzed using a custom bioinformatic pipeline as previously described.18 (link)
Chase A., Pellagatti A., Singh S., Score J., Tapper W.J., Lin F., Hoade Y., Bryant C., Trim N., Yip B.H., Zoi K., Rasi C., Forsberg L.A., Dumanski J.P., Boultwood J, & Cross N.C. (2018). PRR14L mutations are associated with chromosome 22 acquired uniparental disomy, age related clonal hematopoiesis and myeloid neoplasia. Leukemia.
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3

Isolation and Activation of Naïve CD8+ T Cells

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PBMCs were thawed, labeled with CTV and rested overnight. The next day, phenotypically naïve IFNγ producing cells (TVN) and nonproducing (TN) CD8+ T cells were isolated using an IFNγ secretion assay cell enrichment and detection kit (Miltenyi Biotec) and cell sorting. Briefly, cells were transferred onto CD3 coated plates (10 μg/mL; in the presence of soluble CD28 at 5 μg/mL), stimulated for 3 h at 37°C, labeled with IFNγ specific capture antibody reagent and incubated for 45 min at 37°C (while slowly rotating). Next, the cells were concurrently incubated with IFNγ detection antibody and antibodies against the remaining phenotypic T cell markers, which allowed for identification of naive T cell subset. IFNγ producing and nonproducing naïve CD8+ T cell subsets were isolated by cell sorting and incubated in the presence of CD3/CD2/CD28 coated beads (T cell activation/expansion kit, Miltenyi Biotec) at ratio 1:1 in the presence of IL-2 (100 U/mL) for 3 days, when proliferation and phenotype were evaluated by flow cytometry.
Pulko V., Davies J.S., Martinez C., Lanteri M.C., Busch M.P., Diamond M.S., Knox K., Busch E.S., Sims P.A., Sinari S., Billheimer D., Haddad E.K., Murray K.O., Wertheimer A.M, & Nikolich-Žugich J. (2016). Human memory T cells with a naïve phenotype accumulate with aging and respond to persistent viruses. Nature immunology, 17(8), 966-975.
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4

Induction of Regulatory T Cells

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CD4+CD25– T cells were isolated from splenocytes using the CD4+CD25+ regulatory T Cell Isolation Kit (Miltenyi Biotec) as a negative fraction by MACS separator. The purity of the isolated cell population (>95%) was confirmed by flow cytometry.
CD4+CD25– T cells (5 × 105 cells/ml/well) were incubated with vehicle or HDAC6 inhibitor (1 to 10 μM) in the presence of antiCD3/CD28 beads (T cell Activation/Expansion kit, Miltenyi Biotec), and recombinant mouse transforming growth factor (TGF)-β2 (40 pg/ml; R&D systems Inc., Minneapolis, MN, USA) in a 48-well plate (Becton Dickinson) for 6 days at 37 °C in a humidified 5% CO2 incubator.
For surface staining, cells were incubated with PE-Cy7-labeled antimouse CD4 (eBioscience), and APC-labeled antimouse CD25 (eBioscience) for 20 min at room temperature (RT). For intracellular staining, the cells were fixed and permeabilized with fix/permeabilization buffer (eBioscience) for 20 min at 4 °C, washed twice with wash buffer, and then incubated with AlexaFluor488-labeled antimouse forkhead box P3 (Foxp3) and PE-labeled antimouse CTLA-4 (eBioscience) for 20 min at 4 °C in the dark. CTLA-4 expression of induced Foxp3+ Treg cells was analyzed by FACS CantoII flow cytometry (BD bioscience, San Jose, CA, USA) and the results were analyzed with FlowJo software (TreeStar Inc. Ashland, OR, USA).
Oh B.R., Suh D.H., Bae D., Ha N., Choi Y.I., Yoo H.J., Park J.K., Lee E.Y., Lee E.B, & Song Y.W. (2017). Therapeutic effect of a novel histone deacetylase 6 inhibitor, CKD-L, on collagen-induced arthritis in vivo and regulatory T cells in rheumatoid arthritis in vitro. Arthritis Research & Therapy, 19, 154.
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5

Chemotaxis Assay for T and B Cells

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T cells were expanded from PBMCs using the T cell activation/expansion kit (Miltenyi Biotec) and cultured in RPMI containing 10% fetal calf serum, antibiotics, 1 mM glutamine, and recombinant human IL-2 (20 U/ml, R&D Systems) (ThermoFisher). Cells were incubated in Transwell inserts (5 μm, Corning) in 24-well plates containing chemokine (CXCL12 400 ng/ml) for 2 hours at 37°C. The percentage of migrated cells was determined by counting viable (Trypan blue negative) cells in the bottom chamber using the Countess II automated cytometer (ThermoFisher) and dividing by the number of cells in a well without insert. In some experiments, T cells were incubated with tat proteins (400 nM) for 2 hours at 37°C in the presence or absence of forskolin (50 μM) prior to the assay. B cells were incubated in Transwell inserts in wells containing CCL21 at the indicated concentrations for 3 hours at 37°C.
Chinn I.K., Xie Z., Chan E.C., Nagata B.M., Koval A., Chen W.S., Zhang F., Ganesan S., Hong D.N., Suzuki M., Nardone G., Moore I.N., Katanaev V.L., Balazs A.E., Liu C., Lupski J.R., Orange J.S, & Druey K.M. (2021). Short stature and combined immunodeficiency associated with mutations in RGS10. Science signaling, 14(693), eabc1940.
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6

Isolation and Activation of CD8+ T Cells

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The 50 mL heparinized venous blood samples from 10 healthy donors were collected for isolation of PBMCs. CD8+T cells were then isolated from human PBMCs using the CD8 Microbeads (#130-045-201, Miltenyi Biotec), MS columm (Miltenyi Biotec) and MACS Seperator according to the manufacturer’s protocol. For co-culture system, isolated CD8+T cells were activated in T cell activation/expansion kit (Miltenyi Biotec), and then were replated and co-cultured with normal/tumor cells as ratio of 2:1 for 3 days. For IL-10 restimulation experiment, the CD8+T cells were initially split into four groups, control (activated with T cell activation/expansion kit) or activated in addition to different doses of recombinant human IL-10 protein (1 ng/mL, 10 ng/mL and 100 ng/mL) for 3 days. After 3 days, cells were collected, washed and immediately for further flow cytometry.
Qiao M., Zhou F., Liu X., Jiang T., Wang H., Jia Y., Li X., Zhao C., Cheng L., Chen X., Ren S., Liu H, & Zhou C. (2022). Interleukin-10 induces expression of CD39 on CD8+T cells to potentiate anti-PD1 efficacy in EGFR-mutated non-small cell lung cancer. Journal for Immunotherapy of Cancer, 10(12), e005436.
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7

Th17 Cell Induction from Naive CD4+ T Cells

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Naïve CD4+ T cells were cultured in X-Vivo 15 media (Lonza, Cologne, Germany), which combines with 1% human serum and 1% penicillin–streptomycin (Both from Sigma-Aldrich, Saint Louis, USA). For the activation of the T cell receptor (TCR), cells were incubated with the antibodies from T cell Activation/Expansion Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). For the induction of Th17 cells, recombinant human IL-1β (12.5 ng/μl), IL-6 (25 ng/ml; Both from Miltenyi Biotec, Bergisch Gladbach, Germany), recombinant human IL-23 (25 ng/ml; PeproTech, Rocky Hill, USA), and recombinant human TGF-β (25 ng/μl; PAN™-Biotech GmbH, Aidenbach, Germany) were added to cell culture for 96 h as described previously [29 (link)]. To ensure a high induction quality, we changed the medium and cytokines after 72 h of incubation. The induced Th17 cells were collected for quantitative real-time PCR.
Yan S., Golumba-Nagy V., Kotschenreuther K., Thiele J., Refaian N., Shuya D., Gloyer L., Dittrich-Salamon M., Meyer A., Heindl L.M, & Kofler D.M. (2021). Membrane-bound IL-6R is upregulated on Th17 cells and inhibits Treg cell migration by regulating post-translational modification of VASP in autoimmune arthritis. Cellular and Molecular Life Sciences, 79(1), 3.
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8

Generation and Isolation of Th17 Cells

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Generation and isolation of Th17 cells in vitro was done as previously described [13 (link)], [24 (link)]. The isolated Th17 cells were used for coculture experiments with cervical cancer cells in cytotoxicity assays as well as for generation of conditioned media (CM). For CM, cells were cultured at a density of 1 × 106/mL in RPMI1640 medium (Sigma, Taufkirchen, Germany) plus supplements (10% heat‐inactivated endotoxin‐tested FCS (Biochrom, Berlin, Germany) and 1 mm sodium pyruvate) and were stimulated using T‐cell Activation/Expansion Kit (Miltenyi Biotech, Bergisch Gladbach, Germany). After 24 h, CM were collected and analyzed for IL‐17A by DuoSet ELISA (R&D Systems, Minneapolis, USA). Concentrations of IL‐17A in the used CM ranged from 827.4 to 964.9 pg·mL−1.
Theobald L., Stroeder R., Melchior P., Iordache I.I., Tänzer T., Port M., Glombitza B., Marx S., Schub D., Herr C., Hart M., Ludwig N., Meese E., Kim Y., Bohle R.M., Smola S., Rübe C., Solomayer E.F, & Walch‐Rückheim B. (2021). Chemoradiotherapy‐induced increase in Th17 cell frequency in cervical cancer patients is associated with therapy resistance and early relapse. Molecular Oncology, 15(12), 3559-3577.
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9

Differentiation of Human CD4+ T Cells

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Where indicated, CD4+ T cells were cultured in X-Vivo 15 (Lonza, Cologne, Germany) media supplemented with 1% human serum and 1% penicillin-streptomycin (both Sigma-Aldrich, Saint Louis, USA) Cells were cultured for 4 days with the T cell Activation/Expansion Kit (Miltenyi Biotec). For Th0 cell induction recombinant human IL-2 (20 U; Miltenyi Biotech) was added. For Th1 cell induction, recombinant human IL-12 (10 ng/ml; Miltenyi Biotec) and anti-human IL-4 mAb (10 μg/ml; BioLegend) were added. For Th2 cell induction, recombinant human IL-4 (10 ng/ml; PeproTech, Rocky Hill, USA) and anti-human IFNγ mAb (10 μg/ml; BioLegend) were added. For Th9 cell induction, recombinant human IL-4 (10 ng/ml; PeproTech, Rocky Hill, USA), recombinant human TGF-β (5 ng/μl; PAN™-Biotech GmbH, Aidenbach, Germany), and anti-human IFNγ mAb (10 μg/ml; BioLegend) were added. For iTreg cell induction, recombinant human TGF-β (5 ng/μl; PAN™-Biotech GmbH, Aidenbach, Germany) and recombinant human IL-2 (20 U; Miltenyi Biotech) were added as described previously [30 (link)].
Klasen C., Meyer A., Wittekind P.S., Waqué I., Nabhani S, & Kofler D.M. (2019). Prostaglandin receptor EP4 expression by Th17 cells is associated with high disease activity in ankylosing spondylitis. Arthritis Research & Therapy, 21, 159.
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10

Monocyte-T Cell Co-culture for Proliferation

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Monocytes from HCs and patients with cirrhosis expressing high (>20%/450 MFI) versus low (<7.5%/300 MFI) AXL levels were isolated using the Pan Monocyte Isolation Kit (Miltenyi Biotec) and co-cultured with allogeneic CD3+ T cells from a different healthy donor, isolated using the Pan T Cell Isolation Kit (Miltenyi Biotec), in a 1:1 ratio. T cell stimulation was induced with anti-CD2/CD3/CD28 beads (T cell Activation/Expansion Kit; Miltenyi Biotec) as previously described (20 (link)). T cells were stained with carboxyfluorescein succinimidyl ester at day 0. Proliferation was assessed at day 2 of co-culture by flow cytometry.
Brenig R., Pop O.T., Triantafyllou E., Geng A., Singanayagam A., Perez-Shibayama C., Besse L., Cupovic J., Künzler P., Boldanova T., Brand S., Semela D., Duong F.H., Weston C.J., Ludewig B., Heim M.H., Wendon J., Antoniades C.G, & Bernsmeier C. (2019). Expression of AXL receptor tyrosine kinase relates to monocyte dysfunction and severity of cirrhosis. Life Science Alliance, 3(1), e201900465.
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