Immunocult human cd3 cd28 t cell activator

Manufactured by STEMCELL

Sourced in Canada, United States

ImmunoCult Human CD3/CD28 T Cell Activator is a cell culture reagent that activates human T cells. It contains monoclonal antibodies that bind to the CD3 and CD28 receptors on the surface of T cells, triggering their activation and proliferation.

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ImmunoCult Human CD3/CD28/CD2 T Cell Activator (74 citations) Immunocult CD3/CD28 T cell activator (4 citations) Human CD3/CD28 T cell activator (12 citations) ImmunoCult Human T Cell Activator (2 citations) CD3/CD28 T cell activator (12 citations) Immunocult T cell activator (4 citations) Immunocult (16 citations)

98 protocols using immunocult human cd3 cd28 t cell activator

Isolation and Stimulation of Human CD4+ T Cells

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Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats, obtained from healthy donors, by separation on Biocoll Separating Solution (Biochrom, L6115), according to standard procedures. Non-adherent cells were separated from PBMCs after a 30 min adherence step at 37 °C. Next, CD4 + T cells were purified from remaining non-adherent cells using the Human Resting CD4 + T cell Isolation Kit (STEMCELL Technologies, 17962) and following manufacturer´s instructions. For T cell stimulation, we treated CD4 + T cells with either αCD3αCD28 (ImmunoCult™ Human CD3/CD28 T Cell Activator; STEMCELL Technologies, 10971) or IFN I (1:1000, Human IFN Alpha Hybrid (Universal Type I IFN); PBL ASSAY SCIENCE, 11200-1). Cells were cultured in RPMI 1640 (Gibco), supplemented with 10% fetal bovine serum (FBS, Sigma), 20 mM Hepes (Hyclone), 0.3 mg/mL L-glutamine (Hyclone), 100 U/mL penicillin (Gibco) and 100 μg/mL streptomycin (Gibco).
For nucleofection experiments, total human CD4 + T cells were isolated with EasySep Human CD4 + T Cell Isolation Kit (STEMCELL Technologies, 17,952) and cultured in X-VIVO 15 (Lonza). Cells were stimulated with αCD3αCD28 (ImmunoCult™ Human CD3/CD28 T Cell Activator; STEMCELL Technologies, 10971).

Rodríguez-Galán A., Dosil S.G., Hrčková A., Fernández-Messina L., Feketová Z., Pokorná J., Fernández-Delgado I., Camafeita E., Gómez M.J., Ramírez-Huesca M., Gutiérrez-Vázquez C., Sánchez-Cabo F., Vázquez J., Vaňáčová Š, & Sánchez-Madrid F. (2023). ISG20L2: an RNA nuclease regulating T cell activation. Cellular and Molecular Life Sciences, 80(9), 273.

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T Cell Cytotoxicity Assay Protocol

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T cell killing assay was performed as previously described (34 (link)). Human primary T cells (70024, STEMCELL Technologies) were maintained in RPMI-1640 with 10% FBS and 10 ng/mL of IL-2 (589102, BioLegend). For T cell expansion and activation, T cells were cultured in ImmunoCult-XF T Cell Expansion Medium (10981, STEMCELL Technologies) with 25 μL/mL of ImmunoCult Human CD3/CD28 T Cell Activator (10971, STEMCELL Technologies) and 10 ng/mL of IL-2 for 7 days. To analyze the killing effect of T cells, 3 × 10⁵ of tumor cells were cocultured with 3 × 10⁶ of activated T cells in DMEM with 10% FBS, 25 μL/mL of T cell activator and 10 ng/mL of IL-2 for 48 hours. Then LDH release assay was performed.

Wang S., Chang C.W., Huang J., Zeng S., Zhang X., Hung M.C, & Hou J. (2024). Gasdermin C sensitizes tumor cells to PARP inhibitor therapy in cancer models. The Journal of Clinical Investigation, 134(1), e166841.

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Activation of CD4+ T cells by MIAMI cells

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CD4+ T cells (purity > 92%) from 8 different donors were cultured with 20 ng/ml of IL-2 (Roche) and activated using 30 μl of ImmunoCult™ Human CD3/CD28 T Cell Activator (STEMCELL Technologies) per 1 × 10⁶ cells/ml. 2 × 10⁶ CD4+ T cells were plated in the presence or absence of 1 × 10⁵ MIAMI cells. TX or CQ were also added to co-cultures of CD4+ T and MIAMI cells (20:1, respectively). After 3 days of co-culture, CD4+ T cells were collected and stained with Live/Dead-Aqua (Invitrogen), CD4-FITC (BD Bioscience), and activation marker CD25-PE (BD Bioscience) then analyzed by flow cytometry (BD LSRFortessa™ X-20, BD Bioscience) by gating only on live cells.

Rossi F., Noren H., Sarria L., Schiller P.C., Nathanson L, & Beljanski V. (2019). Combination therapies enhance immunoregulatory properties of MIAMI cells. Stem Cell Research & Therapy, 10, 395.

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Isolation and Expansion of CAR-T Cells

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Peripheral blood mononuclear cells (PBMCs) were isolated from cord blood using Lymphoprep (StemCell Technologies, Canada) according to the manufacturer’s instructions. Primary human T cells were isolated from PBMCs via negative selection by using a pan-T Isolation Kit (Miltenyi Biotec, Germany). Isolated T cells were maintained in RPMI-1640 medium supplemented with 10% FBS (Biochrom, Australia), 10 mM HEPES, 100 IU/ml recombinant human IL-2, 2 mM glutamine, and 1% penicillin-streptomycin (Gibco, New York, USA). T cells were stimulated with an ImmunoCult™ Human CD3/CD28 T Cell Activator (StemCell Technologies, Canada) for 48 h. T cells were transfected with CAR vector lentiviral supernatants in the presence of 8 μg/ml polybrene at a multiplicity of infection of 2.0 (Sigma-Aldrich, St Louis, USA). Twelve hours after transfection, T cells were cultured in a fresh medium containing IL-2 (300 U/ml); subsequently, a fresh medium was added every 3 days to maintain cell density within the range of 0.5 to 1 × 10⁶ cells/ml. CAR-T cells determined by flow cytometry at day 5, as GFP+, and then were included in the experiments.

Lin S., Cheng L., Ye W., Li S., Zheng D., Qin L., Wu Q., Long Y., Lin S., Wang S., Huang G., Li P., Yao Y, & Sun X. (2021). Chimeric CTLA4-CD28-CD3z T Cells Potentiate Antitumor Activity Against CD80/CD86–Positive B Cell Malignancies. Frontiers in Immunology, 12, 642528.

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Isolation and Activation of Human CD4+ T Cells

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Human peripheral blood leukopaks (total n=46 different donors) were purchased from either HemaCare (Northridge, CA) (n=15 different donors) or iXSells Biotechnologies (San Diego CA) (n=31 different donors) who collected the leukopaks under either a Hemacare or iXSells Biotechnologies IRB-approved donor consent. Aliquots of the leukopaks were frozen in liquid nitrogen and thawed upon use for CD4 cell isolation. Human primary CD4+ cells were isolated and purified from the leukopak aliquots using a human CD4 T cell negative selection kit (StemCell Technologies). CD4+ cells were maintained in ImmunoCult human T cell expansion medium (StemCell Technologies) with 100 unit/ml human recombinant IL-2 (R&D Systems). To activate CD4+ T cells, 1:40 ImmunoCult human CD3/CD28 T cell activator (StemCell Technologies) was added to the medium, and expanded cells were diluted to 1 million cells per ml at day 3 and day 5 after activation by adding more ImmunoCult human T cell expansion medium with IL-2 (complete medium). CD4+ T cells were cultured for 3–5 days before transfecting with the CBE single base editor mRNA and gRNA.

Weng N., Miller M., Pham A.K., Komor A.C, & Broide D.H. (2021). Single base editing of rs12603332 on Chromosome 17q21 with a Cytosine Base Editor regulates ORMDL3 and ATF6α expression. Allergy, 77(4), 1139-1149.

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Isolation and Culture of Immune Cell Lines

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U937 monocytes and Jurkat T cells were obtained from ATCC (Manassas, VA, USA). The U937 and Jurkat cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS). HEK293T cells were kindly provided by Dr. H. John Sharifi (ACPHS) and were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS. Primary lymphocytes were purchased from the Elutriation Core Facility at University of Nebraska Medical Center. The lymphocytes were cultured with RPMI 1640 supplemented with 5 mM glucose and 10% FBS and were differentiated into T cells with ImmunoCult Human CD3/CD28 T Cell Activator and human IL-2 as per manufacturer’s instructions (Stemcell Technologies Inc.). All cells were cultured at 37 °C and 5% CO₂.

McCaig W.D., Patel P.S., Sosunov S.A., Shakerley N.L., Smiraglia T.A., Craft M.M., Walker K.M., Deragon M.A., Ten V.S, & LaRocca T.J. (2018). Hyperglycemia potentiates a shift from apoptosis to RIP1-dependent necroptosis. Cell Death Discovery, 4, 55.

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Evaluating Allogenic Fibroblast-PBMC Interactions

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Freshly isolated Human or NHP PBMCs were prepared using ACK lysis buffer and labeled with carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen). Subsequently, wild-type (WT) or genetically engineered (GE) fibroblasts were irradiated (with 30 Gy) to stop proliferation. The target fibroblasts were mixed with PBMCs at a 1:1, 10:1, or 100:1 ratio in a 96-well flat-bottom plate for 6 hours to measure SHP-2 phosphorylation, 48 hours to measure intracellular IFN-γ production, or for 6 days to assess proliferation. For signaling experiments, hPBMCs were incubated with ImmunoCult human CD3/CD28 T Cell Activator (Stemcell) and Pansorbin (Millipore Sigma) for 48 hours prior to the addition of porcine fibroblast cells. Cells were labeled with anti-CD3 (Invitrogen), anti-CD4 (Invitrogen), anti-CD8 (Invitrogen), anti-CD16 (Invitrogen), anti-CD20 (Invitrogen), anti-CD28 (Invitrogen), and anti-CD56 (Invitrogen) as per manufacturer instructions. The percentage of positive cells for a given marker were measured by flow cytometry using a BD FACSCanto II cytometer. Data were analyzed using FlowJo software version 10.0 (TreeStar). Assays were performed in triplicate and repeated on three different human or NHP blood donors.

Rao J.S., Hosny N., Kumbha R., Naqvi R.A., Singh A., Swanson Z., Levy H., Matson A.W., Steinhoff M., Forneris N., Walters E., Hering B.J, & Burlak C. (2021). HLA-G1+ Expression in GGTA1KO Pigs Suppresses Human and Monkey Anti-Pig T, B and NK Cell Responses. Frontiers in Immunology, 12, 730545.

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Activation and Stimulation of CD4+ T Cells

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Resting CD4⁺ T cells were labeled with anti-CD4-FiTC antibody (Biolegend, #357406) and isolated from human PBMCs by magnetic activated cell sorting (MACS, Miltenyi) using anti-FiTC microbeads (Miltenyi, Cat#130-048-701) following manufacturer instructions. Then, CD4⁺ T cells were activated for three days with ImmunoCult Human CD3/CD28 T Cell Activator (StemCell, #10971) following manufacturer instructions in the presence of 20 ng/mL IL2 (Novartis, #709421). After activation, cells were expanded for 5 days in the presence of 20ng/mL IL2. Then, cells were starved of IL2 for 24 hours before stimulation as indicated and 10⁵ cells were used per experiment. Cells were attached to coverslips by incubating them at 37°C for 1 hour in PBS, then PBS was replaced with RPMI supplemented with 10% FBS and cells stimulated as described. After stimulation, cells were fixed with 2% formaldehyde for 10 minutes at RT, permeabilized with ice-cold methanol for 20 minutes on ice and stained with anti-STAT3 (Cell Signaling, #9139S) and anti-CDK8 (Invitrogen, #PA1-21780) or anti-STAT3 (Cell Signaling, #9139S) and anti-CDK9 (Cell Signaling, #2316S) for Proximity Ligation Assays following manufacturer instructions (Sigma, #DUO92008).

Martinez-Fabregas J., Wang L., Pohler E., Cozzani A., Wilmes S., Kazemian M., Mitra S, & Moraga I. (2020). CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci. Cell Reports, 33(12), 108545.

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Isolation and Expansion of Human T Cells

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PBMCs were isolated from healthy volunteers as described, and T cells were isolated by immunomagnetic negative selection using EasySep Human T-Cell Isolation Kit (STEMCELL Technologies Inc.). The purified T cells were seeded into 25 cm² flasks at a density of 1×10⁶ cells/mL in 5 mL X-vivo 15 medium supplemented with 10% autologous serum, 100 IU/mL IL-2 and 100 U/mL penicillin/streptomycin, and cultured at 37°C under 5% CO₂. The cells were activated by 25 µL/mL ImmunoCult Human CD3/CD28 T-Cell Activator (STEMCELL Technologies Inc.) for 3 days, and activation of variable CD3⁺ T cells was assessed in terms of surface expression of CD25 using flow cytometry. To expand the T cells, the cell density was adjusted to 1×10⁶ cells/mL with the addition of fresh medium every 2–3 days. For long-term expansion, the cells were harvested and resuspended every 7–10 days in fresh medium and restimulated with ImmunoCult Human CD3/CD28 T-Cell Activator, in addition to adjusting the cell density every 2–3 days, as described. The CD8⁺ T cells were obtained by immunomagnetic positive selection using EasySep human CD8⁺ T-cell Isolation Kit (STEMCELL Technologies Inc.), and purity was verified to be >95%.

Zhang C., Wang Y., Xun X., Wang S., Xiang X., Hu S., Cheng Q., Guo J., Li Z, & Zhu J. (2020). TIGIT Can Exert Immunosuppressive Effects on CD8+ T Cells by the CD155/TIGIT Signaling Pathway for Hepatocellular Carcinoma In Vitro. Journal of Immunotherapy (Hagerstown, Md. : 1997), 43(8), 236-243.

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Cytokine-Induced IFN-γ Production

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Spleens were extracted from mice, homogenized, and treated with ACK RBC lysis buffer to collect mouse splenocytes. Cells were then sorted by FACS using a FACSMelody™ (BD) with antibodies against CD45 (30-F11), CD3Ɛ (145–2C11), CD4 (GK1.5), CD8α (53–6.7), and NK1.1 (PK136) or by MACS using EasySep™ Mouse CD8⁺ or CD4⁺ T Cell Isolation Kits (STEMCELL). 1–2×10⁴ sorted cells were then incubated on 96-well u-bottom plates pre-coated with anti-CDƐ (145–2C11) antibody and cultured in complete RPMI containing 0.01–0.1μg IL-12-Fc or an equivalent volume of PBS. After 48 hours incubation, IFNγ was measured by CBA using Mouse IFNγ Flex Set (558296, BD) according to the manufacturer’s protocol. For human PBMCs, cells were sorted by MACS using EasySep™ Human CD8⁺ (17953, STEMCELL) or CD4⁺ (17952, STEMCELL) T Cell Isolation Kits according to manufacturer’s protocol. 1×10⁴ sorted cells were then incubated on 96-well u-bottom plates in complete RPMI with ImmunoCult™ Human CD3/CD28 T Cell Activator (10971, STEMCELL), adding either 0.1μg of hu-IL-12-Fc or an equivalent volume of PBS. After 48 hours incubation, IFNγ was measured by R&D Human IFN-gamma DuoSet™ ELISA Kit (DY285B, Fisher) according to the manufacturer’s protocol.

Xue D., Moon B., Liao J., Guo J., Zou Z., Han Y., Cao S., Wang Y., Fu Y.X, & Peng H. (2022). A tumor-specific pro-IL-12 activates preexisting cytotoxic T cells to control established tumors. Science immunology, 7(67), eabi6899.

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