Generation of bone marrow-derived MDSCs were prepared according to the previous description [7 (link), 8 (link)]. In brief, bone marrow cells were extracted from the tibias and femurs of mice. Erythrocytes were lysed with ACK Lysis Buffer (Beyotime Biotechnology, C3702-500 ml). Then, the cells were grown in complete RPMI-1640 medium containing 40 ng/ml murine IL-6 (Miltenyi Biotec, 130-096-682) and 40 ng/ml murine GM-CSF (Miltenyi Biotec, 130-095-742) for 4 days. During the 4 days of cell culture, the cells were always placed in an incubator at 37 ℃ and 5% CO2. After 4 days of culture, the cells were removed from the incubator, and the suspended cells were carefully collected, discarding the adherent cells. Follow-up experiments were performed using the collected suspended cells.
Murine il 6
Manufactured by Miltenyi Biotec
Sourced in United States, Macao
Murine IL-6 is a recombinant cytokine that represents the mouse homologue of human interleukin-6. It is a soluble protein that can be used in in vitro cell culture experiments.
Automatically generated - may contain errors
Lab products found in correlation
Human il 2, by Miltenyi Biotec (2 mentions)
Murine gm csf, by Miltenyi Biotec (1 mentions)
Ack lysis buffer, by Beyotime (1 mentions)
Recombinant murine il 2, by Miltenyi Biotec (1 mentions)
Recombinant human il 1β, by R&D Systems (1 mentions)
Tgf β, by R&D Systems (1 mentions)
Il 23, by R&D Systems (1 mentions)
Rpmi 1640, by Miltenyi Biotec (1 mentions)
Gm csf, by Miltenyi Biotec (1 mentions)
Ghost live dead, by Cytek Biosciences (1 mentions)
Non enzymatic solution, by Merck Group (1 mentions)
Cell stimulation cocktail, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Murine il 1β, by Miltenyi Biotec (1 mentions)
Murine il 4, by Miltenyi Biotec (1 mentions)
4 protocols using murine il 6
1
Murine MDSC Generation Protocol
2
Antibody, Cytokine, and Chemical Protocol
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Antibodies are listed in Supplemental Table 2 . Recombinant murine IL-2, human IL-2, and murine IL-6 were purchased from Miltenyi Biotec. Recombinant human IL-1β, IL-23, and TGF-β were purchased from R&D Systems. NECA and CSC were purchased from Merck.
3
Bone Marrow-Derived MDSC Isolation
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BM-MDSCs were cultured as described previously [45] (link). In short, the tibiae and femora were removed from 12-week-old C57BL/6 J male mice to flush BM cells with PBS, after which clean BM cells were resuspended in RPMI 1640 supplemented with 40 ng/mL murine IL-6 and GM-CSF (Miltenyi Biotec, USA).
4
Isolation and Characterization of Murine Immune Cells
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LN and spleen were isolated by dissection from mice and then mashed through a 70-µm filter. Spleen cells were lysed in ammonium-chloride-potassium lysis buffer to remove red blood cells. Cells isolated from spleen and LNs were counted, and 1-5 × 10 6 cells were first stained in PBS and Ghost Live/Dead (Tonbo), followed by blocking in 2.4G2 before staining with the appropriate antibodies for flow cytometry. For transcription factor staining, cells were fixed overnight in the eBioscience Foxp3/Transcription Factor/Fixation-Concentrate kit (Thermo Fisher Scientific). After fixation, cells were permeabilized and stained with the appropriate antibodies. For ICS, cells were stimulated for 4 h in Brefeldin A (eBioscience) and eBioscience Cell Stimulation Cocktail (500×). Cells were then fixed and permeabilized using the BD cytofix/cytoperm kit before staining with the appropriate antibodies.
Islets were purified following standard collagenase protocols as described 22 and dissociated by incubating with a non-enzymatic solution (Sigma, St, Lois, MO) followed by trituration per the manufacturer's instructions.
were purchased from R&D Systems (murine IL-4, murine IL-6, and human IL-2), Miltenyi (murine IL-12, murine IL-1β, and murine IL-23), or HumanKine (human TGFβ).
Islets were purified following standard collagenase protocols as described 22 and dissociated by incubating with a non-enzymatic solution (Sigma, St, Lois, MO) followed by trituration per the manufacturer's instructions.
were purchased from R&D Systems (murine IL-4, murine IL-6, and human IL-2), Miltenyi (murine IL-12, murine IL-1β, and murine IL-23), or HumanKine (human TGFβ).
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