Mouse splenic DCs were isolated using magnetic-activated cell sorting (MACS). Mice were euthanized with CO2, and spleens were removed and placed in spleen dissociation buffer in C tubes (Miltenyi). Spleens were dissociated using homogenizer (Miltenyi) and incubated at 37°C for 15 min. Homogenate was then passed through a 40-µM cell strainer and washed with Dulbecco’s PBS (dPBS). For splenocyte experiments, this single-cell suspension was then cultured for 24 hr in either control RPMI media (150 mmol/L Na+) or media containing 190 mmol/L Na+. To control for hyperosmolality, other cells were exposed to mannitol (190 mmol/L). For DC-specific experiments, DCs were then isolated from this single-cell suspension using the CD11c isolation kit (Miltenyi). For T cell activation experiments, DCs were cultured with T cells (Pan T Cell Isolation Kit II, mouse, 130-095-130) isolated from mice with repeated hypertensive stimuli challenges for 3 days.
Cd11c isolation kit
Manufactured by Miltenyi Biotec
Sourced in France
The CD11c isolation kit is a laboratory equipment product designed for the isolation and purification of CD11c-positive cells from various sample types. It provides a tool for researchers to selectively isolate this cell population for further analysis and experimentation.
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7 protocols using cd11c isolation kit
1
Isolation and Culture of Murine Splenic Dendritic Cells
2
Immunological Response to T. gondii Infection
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Lupus-prone and control mice were infected with T. gondii and 7 days later spleens were removed and splenocytes were prepared. CD11c+ cells were isolated by magnetic separation using positive selection (CD11c+ isolation kit, Miltenyi, Auburn, CA). The flow-through was collected and CD3+ cells were isolated by negative selection from the same animal (Pan T cell isolation kit II, Miltenyi, Auburn, CA). Cells were plated in 96 well plates at a ratio of 5 T cells to 1 dendritic cell (T-3×105: DC-6×104). Antigen was added to the wells (STAg 20 ug/ml) and the plate was incubated for 72 hours at 37°C, 5% CO2. Supernatants were assayed for IFN-gamma production by ELISA (eBioscience, San Diego, CA).
3
Isolation and Co-culture of Dendritic Cell Subsets
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OVA+ migrating LCs and CD11b+ DCs were isolated from sdLN cells by magnetic sorting using a CD11c isolation kit (Miltenyi Biotec, Paris, France) followed by flow cytometry sorting of MHC-II+ CD11c+ OVA+ CD11b+ EPCAM+ and MHC-II+ CD11c+ OVA+ CD11b+ EPCAM−, respectively. As control DCs, MHC-II+ CD11c+ OVA− CD11b− EPCAM− cells were also sorted from the CD11c positive fraction. CD4+ cells were sorted from the CD11c negative fraction using CD4 microbeads (Miltenyi Biotec, Paris, France). Each DC subset was then co-culture with CD4+ cells at ratio 1:5 in 200 μL RPMI 1640 supplemented with FCS, penicillin, streptomycin, and β-mercaptoethanol in 96 wells plate. After 6 days, cells were stained for analysis of Foxp3 or LAP Tregs and expression of CD62L.
4
T Cell Proliferation and IFNγ Production
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For in vitro studies, T cells from OT‐1 Rag−/− mice were isolated using a CD8+ T cell isolation kit (Miltenyi Biotech, Surrey, UK). The purity of responder T cells was assessed using PE‐conjugated anti‐CD8 antibodies (clone 53‐6.7). The purity of T cells was consistently between 90% and 95%. CD11c selected DCs were isolated using a CD11c isolation kit (Miltenyi Biotech) following manufacturers’ instructions. 105 purified CD8+ T cells and 105 CD11c were stimulated in triplicate wells of a 96‐well plate. T cell proliferation was measured by [3H] thymidine incorporation after 3 days in culture. Results are shown as mean count per minute of triplicate determinations ± SD. To measure interferon‐γ (IFNγ) production, culture supernatant, taken from the above cultures, were analyzed using an IFNγ‐specific enzyme‐linked immunosorbent assay (ELISA) kit, following manufacturer's instructions (eBioscience). Results are shown as mean pg/mL of triplicate determinations ± SD.
5
GM-CSF Driven BMDC Generation
6
Isolation and Culture of Murine Splenic Dendritic Cells
7
Isolation and Activation of DCs for NK Cell Co-culture
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DCs were isolated from spleens using a CD11c+ isolation kit (Miltenyi Biotec) as described. In brief, spleen was minced and digested with collagenase D and DNase I at 37°C and filtered through a 70-µM nylon mesh. CD11c+ DCs were then isolated using the manufacturer’s protocol and were activated with 0.5 µg/ml of LPS overnight. After activation, cells were washed with PBS and 1 × 105 DCs were co-cultured with 1 × 105 isolated NK cells in 0.2 ml of RPMI with 10% FBS for 2 d with or without 10 µg/ml of anti–IL-27 or anti–IFN-γ, or the appropriate isotype control antibody. Supernatants were assayed by ELISA (R&D Systems) for IL-27, IFN-γ, or IL-10 production.
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