Bone marrow from naïve mice was cultured for 4 days with IL-6 (40 ng/ml, Peprotech) and GM-CSF (40 ng/ml Peprotech) or media containing 50% tumor-conditioned media, prepared by filtering supernatant from a confluent flask of tumor cells through a 0.45 μm membrane. For suppression assays, MDSCs were either added to 2 × 105 CellTrace-labelled, WT splenocytes simultaneously activated with anti-CD3 (500 ng/ml, clone 2C11, Tonbo) and anti-CD28 (1 μg/ml, clone 37.51, Tonbo) or 2 × 105 CellTrace-labelled, OT-I splenocytes simultaneously activated with OVA257-264 peptide (1 μM, GenScript) in 96-well plates. Proliferation of T cells was measured 3 days later. For chemotaxis assays, MDSCs were separated into Ly6G+ and Ly6G− fractions with anti-Ly6G MicroBeads (Miltenyi) according to the manufacturer’s protocol. Chemotaxis was measured toward recombinant carrier-free osteopontin (R&D Systems) after 1 hr on 3 μm filter plates (Ly6G+ cells) or 4 hrs on 5 μm filter plates (Ly6G- cells) (NeuroProbe).
Clone 37
Manufactured by Cytek Biosciences
Sourced in United States
The Clone 37.51 is a high-performance flow cytometry instrument developed by Cytek Biosciences. It is designed to provide accurate and reliable data analysis for a wide range of applications in cell biology and immunology research. The instrument features advanced optical and fluidic systems that enable precise detection and characterization of cellular populations.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Anti cd3, by Cytek Biosciences (1 mentions)
Ova257 264 peptide, by GenScript (1 mentions)
Anti ly6g microbeads, by Miltenyi Biotec (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Gentamycin, by Thermo Fisher Scientific (1 mentions)
Il 12, by Merck Group (1 mentions)
2 protocols using clone 37
1
MDSC Isolation and Functional Assays
2
Isolation and Culture of CD8+ CTLs
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Inguinal, axillary, brachial, mesenteric, and submandibular lymph nodes (LNs) were excised and T-cells isolated as previously described (Tremblay et al., 2017). Brie y, CD8 + CTLs were enriched by panning and cultured at a density of 1 x 10 6 cells/mL in complete RPMI (cRPMI) media supplemented with CD28 (1 µg/mL; clone 37.51, Tonbo Biosciences, San Diego, CA, USA), gentamycin (5 µg/mL, Gibco), IL-2 (100 U/mL, Peprotech, Rocky Hill NJ, UA), IL-12 (1 ng/mL, MilliporeSigma) in culture dishes that had been precoated overnight with 1 µg/mL CD3 (clone 145-2C11, Tonbo Biosciences) in phosphate buffered saline (PBS, 1X concentration; Corning). cRPMI media was a solution of RPMI 1640 (MilliporeSigma) with 10% FBessence, 100 U/mL of Penicillin/Streptomycin, 55 µM of b-mercaptoethanol, and 10 mM HEPES. Cells were maintained with fresh cRPMI media supplemented with IL-2 (100 U/mL) as required over 7 days. In preparation for labelling, cells were collected from culture dishes, washed with PBS, and re-suspended in cRPMI without antibiotics.
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