α cd28 clone 37

Manufactured by BioLegend

α-CD28 (clone 37.51) is a monoclonal antibody that binds to the CD28 receptor on T cells. CD28 is a co-stimulatory molecule that enhances T cell activation and proliferation when engaged.

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Lab products found in correlation

Cd16 32 fc block, by BioLegend (1 mentions) Cd80 higg1 fc, by BioLegend (1 mentions) Penicillin streptomycin, by Merck Group (1 mentions) Phosflow perm buffer 3, by BD (1 mentions) Phosflow lyse fix buffer, by BD (1 mentions) Easysep mouse cd8 t cell isolation kit, by STEMCELL (1 mentions) Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions) 2 mercaptoethanol, by Merck Group (1 mentions) Mouse il 2, by Thermo Fisher Scientific (1 mentions) αcd3 clone 17a2, by BioXCell (1 mentions) Celltrace cfse, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Clone 37.51 (33 citations) Clone CD28.2 (30 citations) Soluble α-CD28 (clone 37.51; (2 citations)

5 protocols using α cd28 clone 37

CD8+ CTL Activation Assay

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CD8⁺ CTLs (5.0 × 10⁵ cells) sorted from splenocytes were cultured with or without MLACs or MDSCs (4.0 × 10⁵ cells each) in 10% FBS-RPMI containing 0.5 μg/ml α-CD3ε (clone 145-2C11, Biolegend) and 3.0 μg/ml α-CD28 (clone 37.51, Biolegend) in 6-cm dishes. After 72 hr of culture, the cells were harvested and the percentage of activated (CD69⁺) CTL cells within the CD8⁺ cell population was analyzed by flow cytometry.

Tsubaki T., Kadonosono T., Sakurai S., Shiozawa T., Goto T., Sakai S., Kuchimaru T., Sakamoto T., Watanabe H., Kondoh G, & Kizaka-Kondoh S. (2018). Novel adherent CD11b+ Gr-1+ tumor-infiltrating cells initiate an immunosuppressive tumor microenvironment. Oncotarget, 9(13), 11209-11226.

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Comprehensive Immune Cell Phenotyping

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Antibodies against CD8a PECy7 (clone 53-6.7), CD8a APCeFluor780 (clone 53-6.7), CD25 PE (clone PC61.5), CD62L FITC (clone MEL-14), Vα2 PE (clone B20.1) and Vβ5 FITC (clone MR9-4) were obtained from eBioscience. Antibodies against CD44 APC (clone IM7), CD45.1 PECy7 (clone A20), CD45.2 Alexa Fluor 647 (clone 104), CD127 APC (clone A7R34), Ki-67 Alexa fluor 488 (clone 11F6), Bcl-2 PE (clone BCL/10C4), CD16/32 (FcBlock) and activating antibodies α-CD3 (clone 145- 2C11) and α-CD28 (clone 37.51) were obtained from BioLegend.

Rosemblatt M.V., Parra-Tello B., Briceño P., Rivas-Yáñez E., Tucer S., Saavedra-Almarza J., Hörmann P., Martínez B.A., Lladser Á., Rosemblatt M., Cekic C., Bono M.R, & Sauma D. (2021). Ecto-5′-Nucleotidase (CD73) Regulates the Survival of CD8+ T Cells. Frontiers in Cell and Developmental Biology, 9, 647058.

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Quantifying CTLA-4 Blockade by Antibody 4F10

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To determine whether αCTLA-4 clone 4F10 blocks CTLA-4 binding to CD80 and CD86, we permeabilized splenocytes and treated them with graded doses of 4F10 or an isotype control mAb for 60 min at 4°C. All subsequent incubations lasted 30 min at 4°C. Cells were washed, and the amount of residual functionally available CTLA-4 was determined by incubation with 2 μg/ml of recombinant CD80-hIgG1 Fc or CD80-hIgG1 Fc (Biolegend), revealed by subsequent incubation first with 1.25 μg/ml of PE-conjugated goat αhu-man IgG (Southern Biotech) and then with 5 μg/ml PE-conjugated αgoat IgG Abs (Invitrogen). To prevent binding of CD80-hIgG1 Fc or CD80-hIgG1 Fc to CD28 on splenocytes, we blocked CD28 with 10 μg/ml of αCD28 clone 37.51 (Biolegend) during incubation with titrated doses of 4F10. Alternatively to CD80- and CD86-Fc, residual non-blocked CTLA-4 was revealed by incubation with 4 μg/ml APC-conjugated 4F10 mAb (Millipore) under conditions at which displacement of the originally applied blocking antibody is negligible (data not shown). Further staining for CD4 and Foxp3 through conventional protocols identified Treg and Th cells.

Marangoni F., Zhakyp A., Corsini M., Geels S.N., Carrizosa E., Thelen M., Mani V., Prüßmann J.N., Warner R.D., Ozga A.J., Di Pilato M., Othy S, & Mempel T.R. (2021). Expansion of tumor-associated Treg cells upon disruption of a CTLA-4-dependent feedback loop. Cell, 184(15), 3998-4015.e19.

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Quantifying CD8+ T Cell STAT4 Phosphorylation

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Mouse CD8 + T cells were purified from spleens of C57BL/6 mice using EasySep mouse CD8 + T cell isolation kit (Stem Cell). Purified CD8 + T cells (10 6 cells/mL) were activated in six-well plates precoated with 2 μg/mL α-CD3 (clone 17A2, Bioxcell) and supplemented with soluble 5 μg/mL α-CD28 (clone 37.51, BioLegend) and 30 ng/mL mouse IL-2 (Peprotech) for 3 days. Culture medium was IMDM (Gibco) containing 10% heat-inactivated FBS, 1% Penicillin/Streptomycin and 50 μM 2-mercaptoethanol (Sigma Aldrich). After 3 days of culture, activated CD8 + T cells were rested for 6 hrs in fresh culture medium and were transferred into 96-well plates (50,000 cells/well). Indicated amounts of IL-12 or CBD-IL-12 were applied to CD8 + T cells for 20 min at 37 °C to induce STAT4 phosphorylation. Cells were fixed immediately using BD Phosflow Lyse/Fix buffer for 10 min at 37 °C and then permeabilized with BD Phosflow Perm Buffer III for 30 min on ice. Cells were stained with Alexa Fluor (AF) 647-conjugated antibody against pSTAT4 (clone 38, BD) recognizing phosphorylation of Tyr693. Staining was performed for 1 hr at room temperature (RT) in the dark. Cells were acquired on BD LSR and data were analysed using FlowJo (Treestar). Mean Fluorescence Intensity (MFI) of pSTAT4 + population was plotted against cytokine concentration. Dose-response curve was fitted using Prism (v8, GraphPad).

Mansurov A., Ishihara J., Hosseinchi P., Potin L., Marchell T.M., Ishihara A., Williford J.M., Alpar A.T., Raczy M.M., Gray L.T., Swartz M.A, & Hubbell J.A. (2020). Collagen-binding IL-12 enhances tumour inflammation and drives the complete remission of established immunologically cold mouse tumours. Nature biomedical engineering, 4(5).

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Bone Marrow MDSC STING Agonist Proliferation

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Bone marrow MDSCs were generated as above (GM-CSF and IL-6 for 4 days) and treated with STING agonists or vehicle control for 48 hours following CFSE labeling prior to analysis on a flow cytometer. Proliferation index is calculated by the average number of cell divisions that a cell in the original population has undergone. It was obtained by the following formula that measures the ratio of the number of cells that underwent division to the total number of cells in the system.

P I = \sum_{i = 0}^{i} (i * \frac{N_{i}}{2^{i}}) / \sum_{i = 0}^{i} \frac{N_{i}}{2^{i}}

, where i=number of cell division peak as determined by CFSE dilution, N_i=number of cells in that division peak.
For suppression assays, naïve splenocytes were obtained from male C57BL/6 mice, and CD8 T cells were isolated by negative selection with the Miltenyi Mouse CD8 T Cell Isolation Kit. CD8 T cells were labeled with CellTrace CFSE (ThermoFisher) as described. The 1×10⁵ CFSE-labeled CD8 T cells were plated at indicated ratios with unstimulated or repolarized BM-MDSC in 200 µL cDMEM containing 10% FBS, 50 U/mL IL-2, 27.5 µM β-ME, and 2 µg/mL αCD28 (clone 37.51; BioLegend) in round bottom 96-well plates coated with αCD3 (BioXCell clone 145–2 C11; coated overnight at 10 µg/mL in PBS at 4°C). After 72–96 hours, CD8 T cells were analyzed for CFSE dilution by flow cytometry.

Ager C.R., Boda A., Rajapakshe K., Lea S.T., Di Francesco M.E., Jayaprakash P., Slay R.B., Morrow B., Prasad R., Dean M.A., Duffy C.R., Coarfa C., Jones P, & Curran M.A. (2021). High potency STING agonists engage unique myeloid pathways to reverse pancreatic cancer immune privilege. Journal for Immunotherapy of Cancer, 9(8), e003246.

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